150 KDA TGF-B1 accessory receptor acts a negative modulator of TGF-B signaling

ABSTRACT

The present invention relates to a TGF-beta1 binding protein called r150. This protein has a GPI-anchor contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta1 with an affinity comparable to those of the signaling receptors. Furthermore, the released (soluble) form of this protein binds TGF-beta1 independent of the types I and II receptors. Also, the soluble form inhibits the binding of TGF-beta to its receptor. In addition, evidence that r150 is released from the cell surface by an endogenous phospholipase C is provided. Also, the creation of a mutant human keratinocyte cell line with a defect in GPI synthesis which displays reduced expression of r150 is described. Our results using these mutant keratinocytes suggest that the membrane anchored form of r150 is a negative modulator of TGF-beta responses. These findings, taken together with the observation that r150 forms a heteromeric complex with the signaling receptors, suggest that this accessory receptor in either its membrane anchored or soluble form may antagonize TGF-beta responses in human keratinocytes. Experiments with mutants confirmed that TGFbeta1 activity can be modulated when the expression of the accessory receptor r150 is silenced. The complete nucleic acid and deduced amino acid sequences are now provided. The r150 cloned nucleic acid was used to study overexpression of r150. When r150 gene is overexpressed, TGFbeta responses are increased. r150 and its derivatives or precursors (fragments, variants and nucleic acids encoding the same) will find a broad clinical utility, knowing that TGFbeta1 is an important cytokine.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to PCT/CA02/00560 filed on Apr.24, 2002, which claims priority to provisional application No.60/285,713 filed on Apr. 24, 2001, and to provisional application No.60/358,713 filed on Feb. 14, 2002, all of which are hereby incorporatedin their entirety by reference.

BACKGROUND OF THE INVENTION

Transforming growth factor-β (TGF-β) is a 25 kDa multi functional growthfactor which plays a central role in the wound healing process (Robertsand Sporn, 1990; O'Kane and Ferguson, 1997). It is an importantregulator of the immune response (Letterio and Roberts, 1998),angiogenesis, reepithelialization (Roberts and Sporn, 1990),extracallular matrix protein synthesis and remodeling (Peltonen et al,1991; Yamamoto et al, 1994). During wound healing, re-epithelializationinitiates the repair process which is characterized by recruitment ofepidermal stem cells, keratinocyte proliferation and the formation of anepithelial tongue of migrating keratinocytes at the wound edge (Clark,1996). TGF-β is chemotacfic to keratinocytes and induces the expressionof integrins on the migrating epithelium (Helbda, 1988; Zambruno et al,1995). In spite of its promigratory effect on keratinocytes, TGF-β is apotent inhibitor to epithelial cell proliferation in vitro (Pietenpol etat, 1990) and in vivo (Glick et al, 1993). Targeted deletion of theTGF-β1 gene in keratinocytes causes rapid progression to squamous cellcarcinoma (Glick et al, 1994). In addition, the epidermis of transgenicmice expressing a dominant negative TGF-β receptor exhibits ahyperplastic and hyperkeratotic phenotype (Wang et al, 1997). Theseresults support the importance of proper expression of TGF-β andregulation of its function in epidermal development and maintenance ofepidermal homeostasis.

TGF-β is a member of the TGF-β superfamily which also include activins,inhibins, bone morphogenic proteins, growth differentiation factor 1(GDF-1) and glial-derived neurotropic growth factor (GDNF) (Kingsley,1994).

There are three widely distributed TGF-β receptors, type I, type II andtype III, all of which have been cloned (Roberts and Sporn, 1990;Massague, 1998). The types I and II receptors are both transmembraneserine/threonine kinases that are essential for TGF-β signaltransduction. The type III receptor, also known as betaglycan, is a highmolecular weight proteoglycan that is not required for signaling, but isbelieved to play a role in presenting the ligand to the type II receptor(Lopez-Casillas et al, 1993). Endoglin, is another TGF-β receptorpredominantly expressed on endothelial cells (Gougos and Letarte, 1990).According to the present model of TGF-β signal transduction, binding ofTGF-β to the type II receptor which is a constitutively active kinase,leads to the recruitment and phosphorylation of the type I receptor(Wrana et al, 1994). The activated type I kinase phosphorylates thecentral intracellular mediators of TGF-β signalling known as the Smadproteins (Heldin et al, 1997). Smad proteins can be classified intothree groups: the pathway restricted Smads include the Smad2 and Smad3which are phosphorylated by the type I receptor of TGF-β or activin,while the Smads 1, 5 and 8 are implicated in BMP signalling. Thephosphorylation of the pathway restricted Smads permits theirinteraction with the common Smad or Smad4 and this heteromeric complexthen translocates into the nucleus where it regulates expression oftarget genes. Finally, there inhibitory Smads which include the Smad 7and Smad 6 prevent the phosphorlyation of the R-Smads by the type Ikinase. (Heldin et al, 1997, Massague, 1998; Wrana and Attisano, 2000)

In blood circulation, TGF-β1 is found bound to the carrier α₂macroglobulin (α₂M; Webb et al. 1998). α₂M binds many other cytokinesand therefore lacks selectivity for TGF-β1. α₂M polymorphism has beenassociated with Alzheimer's disease, which polymorphism is observed as adeletion in “the bait region” overlapping with TGF-β1 binding domina(Gonias et al. 2000 and Blacker et al 1998).

Although the types I and II receptors are central to TGF-β signaling, itis possible that accessory receptors interacting with the signalingreceptors modify TGF-β responses. For example, both endoglin and typeIII receptor which form heteromeric complexes with the type II receptorhave been reported to modulate TGF-β function. When overexpressed inmyoblasts, endoglin inhibited while type III receptor enhanced TGF-βresponses (Letamendia et al, 1998). In addition, endoglin was shown toantagonize TGF-β mediated growth inhibition of human vascularendothelial cells (Li et al, 2000). Similarly, the newly identified typeI-like receptor BAMBI which associates with TGF-β family receptors caninhibit signaling (Onichtchouk et al, 1999).

There are also a number of molecules that can impact TGF-β signaltransduction by interacting with one or both of the TGF-β signalingreceptors. However, the exact physiological significance of many ofthese interactions are not clearly defined (for review, Massague, 1998).Three of these interacting proteins: the type II TGF-β receptorinteracting protein (TRIP-1) (Chen et al, 1995), Bα (α subunit ofprotein phosphatase A) (Griswold-Prenner, 1998), and serine-threoninekinase receptor associated protein (STRAP) (Datta et al, 1998) allcontain the highly conserved tryptophan-aspartic acid (WD) repeats. WDdomains are important in protein-protein interactions and cellularfunctions such as cell cycle progression and transmembrane signaling(Neer et al, 1994). TRIP-1 is phosphorylated through its interactionwith the type II receptor kinase and exerts an inhibitory effect onTGF-β induced PAI-1 gene transcription, but has no effect on TGF-βmediated growth inhibition (Choy and Derynck, 1998). On the other hand,Bα associates with the type I receptor and positively modulates TGF-βaction. Finally, STRAP can interact with both the type I and IIreceptors and when overexpressed, it exerts an inhibitory effect onTGF-β mediated transcriptional activation. In addition, STRAP can alsointeract with the inhibitory Smad7, but not Smad6. STRAP's interactionwith Smad7 exerts a stabilizing effect on Smad7's association with theactivated type I kinase receptor which prevents Smad2/3's associationand subsequent phosphorylation (Datta and Moses, 2000).

The immunophilin, FKBP12, interacts with the TGF-β type I receptor andacts as a negative modulator of TGF-β function (Wang et al, 1996). Itcan interact with unactivated type I receptor and functions to stabilizethe quiescent receptor state by protecting phosphorylation sites in theGS domain. Upon ligand stimulation, heteromerization and subsequentphosphorylation of the GS domain by the TGF-β type II kinase results inthe release of FKBP12 (Chen et al, 1997; Huse et al, 1999). In contrast,the TGF-β type I receptor associated protein-1 (TRAP-1) interacts onlywith the activated type I receptor kinase (Charng et al, 1998). TRAP-1is not phosphorylated by the type I kinase and TRAP-1's interaction isreported to have an inhibitory effect on TGF-β signaling. However, arecent report describes a different function for TRAP-1 (Wurthner et al,2001). In this study, TRAP-1 was found to associate with inactive TGF-βand activin receptor complexes and upon ligand stimulation, TRAP-1 isreleased. The conformationally altered TRAP-1 is then believed toassociate and subsequently chaperone Smad4 to the activated Smad2. The αsubunit of ras farnesyl protein transferase (FNTA) preferentiallyinteracts with the activated type I receptor and is considered asubstrate because it is phosphorylated by the type I kinase and releasedthereafter (Kawabata et al, 1995). However the functional significanceof this phenomenon remains unexplained. The accessory receptors,endoglin and type III receptor which form heteromeric complexes with thetype II receptor have also been reported to modulate TGF-β function.When overexpressed in myoblasts, endoglin inhibited while type IIIreceptor enhanced TGF-β responses (Letamendia et al, 1998).Glycosylphosphatidyl inositol (GPI)-anchored proteins which lacktransmembrane and cytoplasmic domains have also been shown to bindTGF-β. These proteins have been identified on certain cell lines(Cheifetz and Massague, 1991), but the identity of these GPI-anchoredproteins and the role they may play in TGF-β signaling remain unknown.Recently, the present inventors reported the presence of GPI-anchoredTGF-β binding proteins on early passage human endometrial stromal cells(Dumont et al, 1995), human skin fibroblasts (Tam and Philip, 1998) andkeratinocytes (Tam et al, 1998). On human keratinocytes, they identifieda 150 kDa GPI-anchored TGF-β1 binding protein designated as r150 thatcan form a heteromeric complex with the types I and II TGF-β receptors(Tam et al, 1998). In addition, they demonstrated that upon hydrolysisfom the cell surface by phosphatidylinositol phospholipase C (PIPLC),the soluble form of r150, retains its ability to bind TGF-β1 in theabsence of the types I and II receptors. In addition, it wasdemonstrated that the GPI anchor is contained in a protein with amolecular weight of 150 kDa (Tam et al, 2001). This novel GPI-anchoredTGF-β1 binding protein, r150, has the potential to antagonize orpotentiate TGF-β action in keratinocytes. In the absence of the cDNAencoding r150, one way to examine the effect of r150's loss in TGF-βsignaling is to enzymatically release the binding protein by PIPLCtreatment prior to testing for alterations in TGF-β induced responses.However, the efficacy of exogenously added PIPLC is subject tovariability, being affected by pH, temperature, and acylation ofGPI-anchored proteins (Shukla, 1982; Chen et al, 1998), thus resultsobtained may be difficult to interpret. In addition, GPI-anchoredproteins that are released may get re-synthesized and re-inserted in theplasma membrane soon after PIPLC hydrolysis. Hence, as an alternative,was have created and isolated a keratinocyte cell line that is mutatedin GPI anchor biosynthesis. These cells display a significant loss ofr150 from their cell surface, thus allowing a comparative examination ofTGF-β mediated cellular responses in the GPI anchor deficient cell lineversus the parental HaCat cells under stable experimental conditions

GPI-anchored proteins lack transmembrane and cytoplasmic domains, andare attached to the cell membrane via a lipid anchor in which theprotein is covalently linked to a glycosyl phosphatidylinositol moiety.GPI-anchored proteins have been reported to have roles in intracellularsorting (Rodriguez-Boulan and Powell, 1992), in transmembrane signaling(Brown, 1993) and to associate with cholesterol andglycosphingolipid-rich membrane microdomains (Brown and London, 1998;Hooper, 1999). Also, the GPI anchor enables a protein to be selectivelyreleased from the membrane by phospholipases (Metz et al, 1994; Movahediand Hooper, 1997). r150 was characterized as GPI-anchored, based on itssensitivity to phosphatidylinositol phospholipase C (PIPLC). However, itis important to rule out other possibilities, namely, (i) r150 is notitself GPI-anchored, but is tightly associated with a protein that isGPI-anchored, and therefore is susceptible to release by PIPLC; (ii)r150 is a complex of two lower molecular weight proteins which becameinadvertently cross-linked during the affinity labeling procedure.

It is now demonstrated that the GPI-anchor is contained in r150 itselfand not on a tightly associated protein and that it binds TGF-β1 with anaffinity comparable to those of the signaling receptors. Furthermore,the released (soluble) form of this protein binds TGF-β independent ofthe types I and II receptors. Also, the soluble form inhibits thebinding of TGF-β to its receptor. In addititon, we provide evidence thatr150 is released from the cell surface by an endogenous phospholipase C.Also, a mutant human keratinocyte cell line with a defect in GPIsynthesis was created, which display reduced expression of r150. Theresults using these mutant keratinocytes suggest that the membraneanchored form of r150 is a negative modulator of TGF-beta responses.These findings, taken together with the observation that r150 forms aheteromeric complex with the signaling receptors, suggest that thisaccessory receptor in either its membrane anchored or soluble form andits down- or up-regulation may potentiate or antagonize TGF-β responsesin human keratinocytes, respectively.

The complete amino acid of a molecule named CD109 was recently disclosedas well as the nucleic acids encoding same (Lin et al. 2002). Sequencescomparisons with those of r150 suggest that CD109 is a r150 variant. Nodefinite role has been assigned to CD109 by Lin et al.

SUMMARY OF THE INVENTION

This invention provides a molecule that binds TGF-β1 with a high levelof selectivity. This molecule named r150 can be retrieved in a membraneanchored form or as a released free soluble form. Variants and parts ofr150 which have the property to bind TGF-β1 are grouped under the namer150-like proteins or peptides. They include those defined in SEQ IDNos: 2, 4, 8, 10 and 12. Their corresponding coding nucleic acidsrespectively defined in SEQ ID NOs: 1, 3, 5, 7, 9 and 11.

This invention provides for the use of a protein comprising any one ofthe following sequences in the making of a medication for inhibitingTGF-β1 activity in a biological tissue SEQ ID Nos: 2, 4, 6, 8, 10 and12.

Also provided is the use of an antagonist to a protein comprising anyone of the following sequences in the making of a medication forincreasing TGF-β1 activity in a biological tissue: SEQ ID Nos: 2, 4, 6,8, 10 and 12.

Also provided is the use of a nucleic acid encoding a protein comprisingany one of the following sequences in the making of medication forinhibiting TGF-β1 activity in a biological tissue: SEQ ID Nos: 1, 3, 5,7, 9 and 11.

Also provided is the use of a molecule which silences the expression ofa nucleic encoding a protein comprising any one of the followingsequences in the making of medication for increasing TGF-β1 activity ina biological tissue: SEQ ID Nos: 1, 3, 5, 7, 9 and 11. Particularly, thesilencing molecule is an antisense nucleic acid.

The present inventors being the first to elucidate the complete nucleicacid sequence of r150 and of its deduced amino acid sequence, thisinvention provides an isolated nucleic acid encoding a proteincomprising any one of the following sequences: SEQ ID Nos: 2, 4, 6, 8,10 and 12.

In a specific embodiment, the nucleic acid comprises any one of thefollowing nucleotide sequences: SEQ ID No: 1, 3, 5, 7, 9 and 11.

The nucleic acid is particularly one comprising the nucleotide sequencedefined in SEQ ID No: 1.

The above nucleic acids may include crude nucleic acids or recombinantvectors; namely expression vectors capable of governing transcriptionand translation of the crude nucleic acids inserted downstream to apromotor, are preferred tools for producing r150-like proteins.

Recombinant host cells which comprise the nucleic acids or therecombinant vector are other tools. The vectors are normally selected tocomprise sequences compatible with the host's machinery. Interveningsequences located 5′ and 3′ with regard to the crude nucleic acids areadapted or selected by the skilled artisan desirous to produce aparticular type of host cells. The signal peptide may be charged alsofor another one more appropriate for a given cell type.

There host cells may be domesticated and used in a method of producing ar150-like protein. Such a method comprises the steps of:

-   -   growing a recombinant host cell in a culture medium supporting        cell growth and expression of said nucleic acid:    -   recovering the protein from the culture medium or from the cell.

The nucleic acids may be antisense nucleic acids. They may be insertedin a recombinant vector, namely an expression vector and recombinanthost cells which comprises such antisense nucleic acids can also bemade.

It is further an object of this invention to provide a TGF-β1 bindingreagent, which comprises a protein comprising any one of the followingsequences: SEQ ID Nos 2, 4, 6, 8, 10 and 12.

Compositions of matter which comprise these reagents and a carrier areother objects of this invention.

The carrier may be a pharmaceutical carrier. Otherwise, it may be asolid support to which r150 is bound to immobilize TGF-β1.

DESCRIPTION OF THE INVENTION

r150 is a TGF-β1 binding molecule. Its complete amino acid sequence aswell as the nucleic acid sequence encoding same appear to have beenfirst elucidated by the present inventors. Another group (Lin et al.2002), using a very different approach (affinity binding to monoclonalantibodies) has found a blood cell surface antigen, which they calledCD109. Sequence comparisons show that CD109 (SEQ ID Nos 5 and 6)comprises a 17 amino acid insertion at position 1218–1234 (51 nts). Thisaddition accounts for the difference in amino acids number (1445 forCD109 versus 1428 for r150). Besides that, substitutions of nucleotidesare noted: r150 amino acid thr¹²²⁴ is changed for a methionine (CD109amino acid 1241). CD109 shows polymorphism at residue 703 (Schuh et al.2002). A tyrosine or a serine represent different alleles of CD109. Suchpolymorphism would presumably exist for r150. It is possible that CD109or r150 are responsible for the building of an immune response sinceallo antibodies are retrieved upon administration of CD109 isoforms. Itmay therefore be implied that an isoform compatible with the recipientsubject's tissue may have to be selected as an administrable r150 activeingredient.

r150 binds or sequesters TGF-β1, in its membrane anchored form as wellas in its free soluble form (SEQ ID Nos: 4 and 8). As a result, TGF-β1availability is reduced. The effects induced by TGF-β1 are thereforenegatively modulated (or inhibited). Such inhibition may be desirable inconditions where overproduction of TGF-β1 leads to pathological states(cancer is a specific example of such pathology). On the contrary, inother occasions, increasing TGF-β1 activity may be sought. For example,TGF-β1 encourages tissue or organ graft success. Therefore silencingr150 would have for effect to increase TGF-β1 availability and increasegraft success.

r150 further appears to be related to α₂ macroglobulin (α₂M) and sincethe TGF-β binding domain has been determined for α₂M by Webb et al.(1998), the corresponding domain in r150 is presumed to be located aregion corresponding to α₂ macroglobulin amino acids 666–706. Thesecorresponds to r150 amino acids 651–683 (SEQ ID No: 10). Therefore, ther150 peptide having the sequence defined in SEQ ID NO: 10 is alsocontemplated as TGF-β1 binding peptide within the scope of theinvention; the nucleic acid encoding this peptide is as well.

Webb et al. (2000) even found the minimal α₂M TGF-β1 binding sequencewhich appears to be delineated by amino acid 717 and 733. Thecorresponding strectch in r150 is found between amino acid residues 694and 712 (SEQ ID No.12).

Gomas et al. (2000) reported that α₂M gene polymorphism has beenassociated with Alzheimer's disease, which polymorphism is observed as adeletion in “the bait region” overlapping with TGF-β1 binding domain. Itis envisageable that r150 could be useful to sequester and neutralizeTGF-β1 especially in diseases wherein α₂M is deficient. Any portion ofr150 or variants thereof that is capable of binding TGF-β1 activity isintended to be used in the making of a medication or a method orcomposition for inhibiting TGF-β1 activity. This includes proteins orpeptides comprising sequences defined in SEQ ID NOs. 2, 4, 6, 8, 10 and12. These r150-like proteins or peptides would include any moleculehaving at least 50% homology with the above sequences. On the opposite,any molecule having an antagonistic activity to the above r150-likeproteins or peptides would find a use in the making of a medication or amethod or a composition for increasing TGF-β1 activity.

Nucleic acids encoding the above r150-like proteins or peptidesrepresent an alternative to the direct administration of proteins orpeptides. Antisense nucleics would on the opposite silence theexpression of r150-like proteins or peptides. All these nucleic acidsinclude recombinants vectors, namely expression vectors, which areavailable and well known to the skilled artisan.

A very large body of literature describes diseases or disease modelsinvolving up and down regulation of TGF-β1 activity. Nowadays, TGF-β1binding proteins decorin and an anti-TGF antibody are currently underclinical trials. The present r150-like proteins or peptides couldrepresent a valuable and advantageous alternative to these molecules,because of their selectivity for TGF-β1 isoform, combined to theirhydrosolubility.

Here is a non-exhaustive list of disease models where alteration ofTGF-β action has been shown to be of therapeutic benefit:

Cancer progression:

Note: TGF-β has biphasic effects during tumorigenesis, acting early as atumor suppressor, but later stimulating cancer progression.

(i) Suppression of tumor progression by TGF-β

Akhurst R. J. and Derynck R. (2001). TGF-β signaling in cancer adouble-edged sward. TRENDS in Cell Biology 11: S44–S51.

Welch, Dr. et al (1990). Transforming growth factor-β stimulates mammaryadenocarcinoma cell invasion and metastatic potential. Proc. Natl. Acd.Sci. USA 87: 7678–7682

Markowitz, S. et al. (1995) Inactivation of the type II TGF-β receptorin colon cancer cells with microsatellite instability. Science268,1336–1338.

Massague .J. et al. (2000) TGF-β signaling in growth control, cancer,and heritable disorders. Cell 103, 295–309.

(ii) Stimulation of tumor progression by TGF-β

Hojo, M. et al. (1999) Cyclosporine induces cancer progression by acell-autonomous mechanism. Nature 397, 530–534.

Yin, J. J. et al. (1999) TGF-β signaling blockade inhibits PTHrPsecretion by breast cancer cells and bone metastases development. J.Clin. Invest. 103, 197–206.

Exogenous TGF-β1 promotes wound healing where as inhibiting TGF-β1activity or enhancing TGF-β3 activity reduces scarring in animal models

Roberts, A. B., and Sporn, M. B. (1996). Transforming growth factor R.A. F. (ed), Plenum Press, New York, p275–308.

Mustoe, T. A., Pierce, G. F., Thomason, A., Gramates, P., Sporn, M. B.,and Deuel, T. F. (1987). Accelerated healing of incisional wounds inrats induced by transforming growth factor-β. Science 237: 1333–1336.

Quaglino, D., Nanney, L. B., Ditesheim, J. A., and Davidson, J. M.(1991). Transforming growth factor-β stimulates wound healing andmodulates extracellular matrix gene expression in pig skin: incisionalwound model. J. Invest. Dermatol. 97: 34–42.

O'Kane, S., and Ferguson, M. W. J. (1997). Transforming growth factor-βsand wound healing. Int. J. Biochem. Cell. Biol. 29:63–78.

Shah, M., Foreman, D. M., and Ferguson, M. W. J. (1995). Neutralizationof TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous ratwounds reduces scarring. J. Cell Science 108: 985–1002.

Choi, B-M., Kwak, H-J., Jun, C-D., Park, S-D., Kim, K-Y., Kim, H-R., andChung, H-T. (1996). Control of scarring in adult wounds using antisensetransforming growth factor-β1 oligodeoxynucleotides. Immunol. Cell Biol.74:144–150.

Blocking TGF-β1 overproduction reduce tissue fibrosis (pulmonaryfibrosis, liver cirhosis, glomerulonephritis, scleroderma andatherosclerosis).

Border et al, (1990). Suppression of experimental glomerulonephritis byantiserum against transforming growth factor beta 1. Nature 346 (6282):371–374

Isaka Y., Brees D. K., lkegaya K., Kaneda Y., Imai E., Noble N. A.,Border W. A. (1998). Gene therapy by skeletal muscle expression ofdecorin prevents fibrotic disease in rat kidney. Nat. Med. 2: 418–423.

Isaka Y., Akagi Y., Ando Y., Tsujie M., Sudo T., Ohno N., Border W. A.,Noble N. A., Kaneda Y., Hori M., and Imai E. (1999). Gene therapy bytransforming growth factor-beta receptor-IgG Fc chimera suppressedextracellular matrix accumulation in experimental glomerulonephritis[see comments]. Kidney Int. 55:465–475.

Khalil, N. and Greenberg A H (1991). The role of TGF-β in pulmonaryfibrosis. Ciba Found Symp. 157: 194–207.

Yamamoto, T., Takagawa S., Katayama I., and Nishioka K. (1999).Anti-Sclerotic effect of transforming growth factor-beta antibody in amouse model of bleomycin-induced scleroderma. Clin Immunol, 92(1):6–13.

Gressner, A. M., Weiskirchen, R., Breitkopf K., and Dooley S. (2002).Roles of TGF-beta in hepatic fibrosis. Front Biosci (7): d793–807.

Sheppard D. (2001). Integrin-mediated activation of transforming growthfactor-beta(1) in pulmonary fibrosis. Chest 120(1 Suppl):49S-53S.

McCaffrey, T. A. (2000). TGF-βs and TGF-β receptors in atherosclerosis.Cytokine. Growth Factor Rev. 11: 103–114.

TGF-beta has tissue protective effects (against ischemia reperfusioninjury) in the heart, brain and kidney.

Lefer A M., Ma X-L, Weyrich A S, Scalia R. (1993). Mechanism of thecardioprotective effect of TGF-β1 in feline myocardial ischemia andreperfusion. Proc. Natl. Acad. Sci. USA 90: 1018–1022.

Lefer A M, Tsao P, Aoki N, Palladino M A. (1990). Mediation ofcardioprotection by transforming growth factor-β. Science 249: 61–64.

McNeill H. Williams C, Guan J, Dragunow M, Lawlor P, Sirimanne E,Nikolics K,

Gluckman P. (1994). Neuronal rescue with transforming growth factor-beta1 after hypoxic-ischaemio brain injury. Neuroreport 5: 901–904.

Mehta J L, Yang B C, Strates B S, Mehta P. (1999). Role of TGF-beta1 inplatelet-mediated cardioprotection during ischemia-reperfusion inisolated rat hearts. Growth Factors 16: 179–190.

Recombinant hosts comprising the above nucleic acids or recombinantexpression vectors can be used as a biological machinery in theproduction of the r150-like proteins or peptides. The elucidation of thenucleic acid sequence of r150 therefore leads to a method of producingthese proteins or peptides by recombinant technology.

A variety of TGF-β1 binding reagents and compositions may be derivedfrom the present invention.

First, peptides such as those defined in SEQ ID. Nos: 10 and 12 may beused as such as a TGF-β1 binding reagent. Larger molecules like thosedefined in SEQ ID Nos 2, 4, 6 and 8 could be conjugated (through theiranchoring region) to a carrier. The carrier may take the form, forexample, of a solid or semi-solid medium (beads, chromatography columns,plates, etc.), to immobilize TGF-β1. Pharmaceutical compositions wouldtake any suitable form, depending on the selected route ofadministration. A r150-like protein or peptide (SEQ ID Nos: 2, 4, 6, 8,10 and 12) would be formulated with a pharmaceutically acceptablecarrier. Doses equivalents those used by intravenous route for decorinand/or the TGF-antibody can be produced.

Objects, advantages and features of the present invention will becomemore apparent upon reading of the following non restrictive descriptionof preferred embodiments thereof, given by way of example only withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Affinity cross-link labeling of human neonatal keratinocyteswith ¹²⁵I-TGF-β1. Confluent monolayers were affinity labeled with 100 pM¹²⁵I-TGF-β1 in the absence or presence of unlabeled TGF-β1, -β2, or -β3.Solubilized cell extracts were analyzed by SDS-PAGE under non reducingconditions and autoradiography (a). Competition curves for r150 and thetypes I and II TGF-βreceptors were derived by densitometric analysis ofa typical autoradiogram. The data for each binding complex are expressedas a percent of the value in control wells incubated with ¹²⁵I-TGF-β1alone and are plotted against the concentration of unlabeled TGF-β1 (b),-β2 (c), or -β3 (d). The autoradiogram and competition curves arerepresentative of three different experiments.

FIG. 2: Temperature induced phase separation in Triton X-114 of TGF-βbinding proteins on human neonatal keratinocytes. Keratinocytes affinitylabeled with ¹²⁵I-TGF-β1 were lysed in 1% Triton X-114. The Triton X-114soluble material was incubated at 30° C. for 10 minutes followed bycentrifugation at room temperature to induce phase separation of thedetergent rich phase and the aqueous detergent poor phase. A: An aliquot(20%) from each phase was precipitated with ethanol/acetone, andanalyzed by SDS-PAGE under reducing conditions. ¹²⁵I-TGF-β1 labeledproteins in the aqueous (Aq) and the detergent rich (Det) phases,representative of three different experiments, are shown. B: Theremaining 80% of the detergent phase was utilized to determine theeffect of PIPLC treatment on the partitioning of r150 in Triton X-114.The detergent phase was incubated in the absence (−) or presence (+) of0.6 U/ml of PIPLC followed by temperature induced phase separation andethanol/acetone precipitation as above, to distinguish between thehydrophilic and amphipathic forms of the proteins. Analysis of the¹²⁵I-TGF-β1 labeled proteins in the aqueous phases of PIPLC treated (+)and untreated (−) samples, by SDS-PAGE under reducing conditions areshown. The results shown are representative of two differentexperiments.

FIG. 3: (a) Affinity labeling of soluble r150 with ¹²⁵I-TGF-β1. Toverify that the soluble form of the r150 can bind to TGF-β1, humankeratinocytes (HaCaT) were left untreated (−) or treated with 0.6 U/mlof PIPLC (+). The GPI-anchored proteins released into the supernatantwere concentrated and an aliquot was affinity cross-link labeled with150 pM of ¹²⁵I-TGF-β1 in the absence or presence of excess unlabeledTGF-β1 and subjected to SDS-PAGE under reducing conditions. The resultshown is representative of four different experiments. (b) inhibition of¹²⁵I-TGF-β1 binding to TGFβ receptors by soluble r150. Confluentmonolayers of HaCaT cells grown in T-25 cm² culture flasks were leftuntreated or treated with 0.6 U/ml of PIPLC for 60 minutes at 37° C. Thesupernatants were collected and concentrated by Centricon. MvLu1 cellswere affinity labeled with 50 pM ¹²⁵I-TGF-β1 in the absence or presenceof indicated doses of supernatants and ¹²⁵l-TGF-β1 specifically boundwas plotted as a function of the amount of supernatant used. Thearbitrary unit of “1” is equivalent to a dose of supernatant from a T-25cm² flask (approximately 1×10⁶ cells). (c) Effect of anti-TGF-β1 on theinhibition of TGF-β1 binding to TGF-β receptors. Confluent monolayers ofMvLu1 cells were affinity labeled with 50 pM ¹²⁵I-TGF-β1 in the absence(C), or presence of PIPLC treated supernatant (+PIPLC-S), or PIPLC-Spretreated with non-immune rabbit IgG (15 μg/ml), or PIPLC-S pretreatedwith anti-TGF-β1 antibody (15 μg/ml). To demonstrate that theanti-TGF-β1 antibody effectively neutralizes TGF-β, experiments werealso performed with 100 pM of TGF-β1 (+β1), β1 pretreated withnon-immune rabbit IgG (15 μg/ml), or β1 pretreated with anti-TGF-β1antibody (15 μg/ml). The values shown in (b) and (c) are the mean(+/−S.D.) of at least three to five different experiments.

FIG. 4: Identification of a GPI-anchor in r150. Human neonatalkeratinocytes were harvested and treated with PIPLC (as described inMaterials and Methods). The supernatant containing the GPI-anchoredproteins were purified using a TGF-β1 affinity column (see Materials andMethods for details). After the addition of the sample to the column,0.5 ml fractions were collected during washing and elution. (a) Analiquot from each fraction was affinity labeled with 150 pM of¹²⁵I-TGF-β1 in the absence or presence of excess unlabeled TGF-β1 andsamples were analyzed by SDS-PAGE under reducing conditions. Onlyfraction 21 demonstrated an affinity labeled protein at 150 kDa while inadjacent fractions no 150 kDa band was detectable. Affinity labelingpattern obtained for fraction 21 and fraction 18 are shown. (b) Selectedfractions were subjected to SDS-PAGE and transferred to a nitrocellulosemembrane, and the samples were immunoblotted with an anti-CRD antibody(Oxford GlycoSystems). A 150 kDa protein was detected in fraction 21 butnot in adjacent fractions (fraction 18). Immunoblotting with theanti-CRD antibody was performed twice and the affinity cross-linklabeling experiments of soluble r150 was done at least three times.

FIG. 5: Immunoprecipitation of affinity labeled TGF-β binding complexeson human neonatal keratinocytes with the anti-CRD antibodies.Keratinocytes not treated with PIPLC were affinity labeled with 100 pM¹²⁵I-TGF-β1 (a & b) or ¹²⁵I-TGF-β2 (c) and were not immunoprecipitated(nip) or subjected to immunoprecipitation with an anti-CRD antibodyagainst trypanosomal sVSG (Oxford GlycoSystems) (a & c), or with ananti-CRD antibody against porcine membrane dipeptidase (Broomfield andHooper, 1993) (c). In the lane marked anti-CRD+peptide theimmunoprecipitation was carried out using the anti-CRD antibody whichwas proincubated with PIPLC treated membrane dipeptidase. Immunecomplexes were subjected to SDS-PAGE under reducing conditions andanalyzed by autoradiography. The results shown are representative of atleast four to five experiments.

FIG. 6A: Expression of CD59 is decreased in keratinocytes mutated in GPIanchor biosynthesis. A representative histogram of the expression ofCD59 in a GPI anchor deficient clone, GPI M (white columns) and parentalHaCat cells (black columns) as assessed by flow cytometry using ananti-CD59-FITC labeled antibody. The immunoflourescence intensity ofCD59 in the GPI anchor deficient cells was approximately 50% to that ofthe HaCat cells. The control performed in the absence of antibody isalso included (doted peak). Flow cytometry was performed at least threetimes.

FIG. 6B: Cell surface expression of r150 is markedly decreased in GPIanchor mutated cells. Confluent monolayers of HaCat and GPI M cells wereaffinity labeled with 100 pM ¹²⁵I-TGF-β1. Solubilized cell extracts wereanalyzed on SDS-PAGE under reducing conditions.

FIG. 7A: Doubling time curves of HaCat and GPI anchor mutated cells.HaCat and GPI M cells were seeded at 8.0×10⁵ cells in 60 mm dishes induplicate and the cell number for each was determined at 14, 24 28, 32,36, 48, 52 and 72 hours using a heamacytometer.

FIGS. 7B and C: The cellular morphology of GPI anchor mutated cells isidentical to that of HaCat cells. Microscopic representation (10×magnification) of HaCat (FIG. 7B) or GPI M cells (FIG. 7C) under normalculture conditions.

FIGS. 8A and B: Enhanced TGF-β stimulated transcriptional response inGPI anchor mutated cells. HaCat and GPI M cells were transientlytransfected with 1 μg of p3TP-Lux reporter gene construct and were leftuntreated or treated under serum free conditions with the indicatedconcentrations of TGF-β1 for 4 hrs (FIG. 8A) or 16 hrs (FIG. 8B). Theluciferase activity was normalized to β-galactosidase activity expressedfrom a co-transfected CMVβgal plasmid. The data is representative of atleast three independent experiments. Error bars represent standarddeviation.

FIGS. 9A and B: GPI anchor mutated cells display enhanced responses atlow doses of TGF-β1. HaCat and GPI M cells were left untreated ortreated with the indicated doses of TGF-β1 (FIG. 9A) and TGF-β2 (FIG.9B) under serum free conditions for 20 minutes. Immunoblotting wasperformed using a rabbit polyclonal antibody specific to thephosphorylated form of Smad2. Immmunoblotting was repeated with ananti-Smad2 antibody that recognizes total Smad2 (unphosphorylated andphosphorylated forms) or an anti-STAT3 antibody to demonstrate equalprotein loading. This data is representative of three differentexperiments.

FIGS. 10A, B and C: Elevated Smad2 phosphorylation is sustained in GPIanchor mutated cells. HaCat and two GPI anchor deficient clones GPI M(FIG. 10A) and GPI M1 (FIG. 10B) or a keratinocyte clone that was notGPI anchor mutated, GPI NM (FIG. 10C) were treated with 100 pM TGF-β1for the indicated times. The control lane (−) received no TGF-β1treatment. Imnmunoblotting was performed using a rabbit polyclonalantibody specific to the phosphorylated form of Smad2. The samenitrocellulose membrane was reblotted and with an anti-STAT3 antibody todemonstrate equal protein loading. This data is repesentative of atleast three different experiments.

FIG. 11: Autophosphorylation of type II kinase in HaCat and GPI anchormutated cells. HaCat or GPI M cells were left untreated (−) or treated(+) with 100 pM of TGF-β1 for 20 minutes. Precleared lysates wereimmunoprecipitated with 3 μg/ml of anti-type II receptor antibodyovernight. Following adsorption to protein A sepharose beads, 10 μCi ofgamma ³²P was added to the immunocomplexes and incubated for 30 minutesat 30° C. to allow phosphorylation to occur. The reaction was halted bythe addition of sample buffer and immunocomplexes were then subjected toSDS-PAGE under reducing conditions. This data is representative of twodifferent experiments.

FIG. 12: Schematic diagram representing the cloned sequence of the r150protein.

FIG. 13: HaCaT or 293 cells were transfected with r150 gene or the emptyvector (pCMV sport 6) and cell lysates were fractionated by SDS-PAGE andtransferred onto nitrocellulose membrane and immunoblotted with anti-CRDantibody. The Western blot shown is representative of four experiments(two each with HaCaT and 293 cells).

FIG. 14: HaCaT or 293 cells were transfected with r150 gene or the emptyvector (pCMV sport 6) or were left untransfected. Cells were allowed torecover for 24 hrs and were treated with 100 pM TGF-1 for 30 minutes.Cell lysates were then Western blotted with an anti-phosphoSmad2antibody.

FIG. 15: HaCaT or 293 cells were transfected with r150 gene or the emptyvector (pCMV sport 6) or were left untransfected. Cells were allowed torecover for 24 hrs and were treated with 100 pM TGF-1 for 24 hours, orwere left untreated. The luciferase activity was normalized toglactosidase activity obtained from a cotransfected CMV gal palsmid.

FIG. 16: Schematic model of the mechanism by which r150 inhibitsTGF-responses.

FIG. 17: Sequences of CD109, publishes by another, represent r150putative variants. (Genbak accession numbers AF410459 and AAL84159.1.)

FIG. 18: Alignment of α₂-macroglobulin and r150 partial sequences.

EXAMPLE 1 Characterization of TGF-β1 Binding Protien Different fromother TGF-β Receptors Methods

Cell Culture:

Neonatal keratinocytes were prepared from foreskin tissue obtained atnewborn male circumcision as described by Germain et al (1993). Thekeratinocytes were cultured in keratinocyte serum free medium (Gibco,Burlington, Ontario) and cells of third to fifth passage were used forexperiments. The immortalized keratinocyte cell line, HaCaT, wasobtained from Dr. Boukamp (Heidelberg, Germany), and the mink lungepithelial cells (Mv1Lu) were from ATCC. Both cell types were maintainedin Dulbecco's Minimal Essential Medium (D-MEM) supplemented with 5% FBS,1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 0.25 μg/ml amphotericin (Gibco, Burlington, Ontario).All cells were maintained at 37° C. in an atmosphere of 5% CO₂/air.

Affinity Labeling of Cells:

Iodination of TGF-β1 (Collaborative Biomedical) was done as described(Philip and O'Connor-McCourt, 1991). Affinity labeling technique wasperformed as detailed previously (Dumont et al, 1995). Briefly, cellmonolayers were washed with ice-cold binding buffer (D-PBS or Dulbecco'sphosphate-buffered saline with Ca²⁺ and Mg²⁺, pH 7.4) containing 0.1%bovine serum albumin (BSA). Cells were incubated with 100–200 pM of¹²⁵I-TGF-β1 for three hours at 4° C. In some experiments, incubationswere done in the absence or presence of increasing concentrations ofunlabeled TGF-β isoforms to determine the competition profiles of thereceptors. The receptor-ligand complexes were cross-linked with 1 mMBis-(Sulfosuccinimidyl) suberate (BS³, Pierce). The reaction was stoppedby the addition of glycine and the cells were solubilized, and separatedon 3–11% polyacrylamide SDS gel. The results were analyzed by usingautoradiography followed by quantitative densitometry (Gel-Cypher,Lightools Inc, Encinitas, Calif. or ImageQuant, Molecular Dynamics,Sunnyvale, Calif.)

Temperature Induced Phase Separation in Triton X-114 of r150 andHydrolysis by PIPLC:

Temperature induced phase separation in Triton X-114 and PIPLC treatmentwas performed as described previously with modifications (Bordier,1981). Keratinocytes were affinity labeled with 150 pM of ¹²⁵I-TGF-β1and lysed in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1%Triton X-114, 1 mM phenylmethylsufonyl fluoride and protease inhibitorcocktail (200 μg/ml BSA, 1 μg/ml leupeptin, 10 μg/ml benzamide, 10 μg/mlsoyabean trypsin inhibitor and 2 μg/ml pepstatin) for 60 minutes at 4°C. The cell lysates were centrifuged at 13 000×g for 15 minutes at 4° C.The Triton X-114 soluble material was incubated at 30° C. for 10 minutesfollowed by a 10 minute centrifugation at 13 000×g at room temperatureto separate the detergent rich phase from the aqueous detergent poorphase. An aliquot (20%) from each phase was precipitated withethanol/acetone, and analyzed by SDS-PAGE and autoradiography. Theremaining 80% of the detergent phase was utilized to determine theeffect of PIPLC on the detergent solubility of r150 using the method ofLisanti et al (1988). Briefly, the GPI-anchored protein enricheddetergent phase was incubated with or without 0.6 U/ml of PIPLC (RocheDiagnostics) for one hour at 37° C. with mild agitation. Temperatureinduced phase separation was then repeated. Both the aqueous anddetergent phases were precipitated by adding ethanol/acetone, andsubjected to SDS-PAGE and autoradiography.

Affinity Labeling of Soluble r150:

Neonatal keratinocytes were harvested by treating confluent monolayerswith Hanks' balanced salt solution containing 5 mM EDTA (pH 7.5). Thecell pellet was washed with D-PBS and treated with 0.6 U/ml of PIPLC orleft untreated, for one hour at 37° C. with mild agitation.

The supernatant containing the released GPI-anchored proteins wascollected and concentrated by Centricon 30 (Amicon). Aliquots of theconcentrated supernatant were affinity labeled with 150 pM of¹²⁵I-TGF-β1 in the absence or presence of excess unlabeled TGF-β (7.5nM) and analyzed by SDS-PAGE as described above except that thesolubilization step was omitted.

¹²⁵I-TGF-β1 Binding to Mv1Lu Cells

To test whether soluble r150 regulates the binding of TGF-β to itsreceptors, the supernatant obtained from PIPLC treated HaCaT cells wereused in a ¹²⁵I-TGF-β1 Mv1Lu binding assay. The HaCaT cells; whichdisplay the r150 with identical properties as the neonatal keratinocytes(Tam et al, 1998), were grown in T-25 cm²tissue culture flasks (Falcon)were left untreated or treated with PIPLC, and the resultingsupernatants were concentrated by Centricon 30 (Amicon). Mv1Lu cellswere incubated with 50 pM of ¹²⁵I-TGF-β1 in the absence or presence ofincreasing doses of the concentrated supernatant for three hours at 4°C. The cells were washed, solubilized and the bound radioactivity wasdetermined by a gamma counter.

To rule out the possibility that any alteration in ¹²⁵I-TGF-β1 bindingcaused by the supernatants was not due to the presence of TGF-β1, theabove binding assay was also done in the presence of the supernatanttreated with an anti-TGF-β1 antibody. The supernatant was incubated withthe antibody (15 μg/ml) overnight at 4° C. It was then precleared ofimmune complexes and excess antibody that may interfere with the assay,by incubating with a protein A Sepharose slurry (PharmaciaBiotech) fortwo hours before addition to the assay. The TGF-β1 antiserum (obtainedfrom Dr. M. O'Connor-McCourt; Moulin et al, 1997) was purified on aHiTrap Protein G column (PharmaciaBiotech) following standardprocedures. The specificity of the antibody was verified by Western Blotanalysis

Determination of TGF-β Concentration in HaCaT Supernatant:

The concentration of TGF-β1 was quantitated in the supernatants fromHacaT calls left untreated or treated with PIPLC using a mink lungepithelial cells-luciferase assay as described by Nunes et al (1996).This quantitative bioassay for TGF-β is based on the ability of TGF-β toinduce the expression of plasminogen activator inhibitor type 1 (PAI-1)gene. The mink lung epithelial cells stably transfected with anexpression construct containing a truncated PAI-1 promoter fused to theluciferase reporter gene were provided by Dr. D. B. Rifkin (New YorkUniversity Medical Center). Briefly, these cells were incubated withvarying doses of HaCaT supernatant, and the luciferase activity(expressed as relative light units) was quantitated by a BertholdLuminometer. Recombinant TGF-β1 (Austral Biochemicals; 0.5 pM-50 pM) wasused to create a standard curve.

Immunoaffinity Chromatography and Immunoblotting of r150:

Neonatal keratinocytes were harvested by treating confluent monolayerswith Hanks' balanced salt solution containing 5 mM EDTA (pH 7.5). Thecell pellet was washed with D-PBS and treated with 0.6 U/ml of PIPLC forone hour at 37° C. with mild agitation. The supernatant containing thereleased GPI-anchored proteins was purified through a TGF-β1 affinitycolumn (made available by Dr. M. O'Connor-McCourt, Montreal, Quebec).The column was prepared by incubating one milligram of TGF-β1 in a 200mM HCO₃ (pH 8.3) buffer containing 30% (v/v) of n-propanol with fivemilligrams of Reacti-gel (Pierce) for 72 hours at 4° C. The reaction wasstopped by the addition of 200 μl of 2 M TBS (pH 7.4), and the gel waswashed to remove any unbound TGF-β1. The supernatant containing theGPI-anchored medium was loaded on the column equilibrated with 60 mMTris (pH 7.4) and eluted with a 10 mM citrate/300 mM NaCl buffer (pH2.5). Fractions of 0.5 ml were collected, and each fraction was analyzedfor binding to TGF-β1, and immunoblotted for the presence of GPI anchor.

To verify binding to TGF-β1, an aliquot from each fraction was affinitylabeled with 150 pM of ¹²⁵I-TGF-β1 and analyzed by SDS-PAGE andautoradiography as described above, except that the solubilization stepwas omitted.

The fraction containing r150 and adjacent fractions were immunoblottedwith the anti-CRD antibody to detect the GPI anchor. The anti-CRDantibody is specific to the inositol 1,2 cyclic monophosphate moiety,known as the “cross-reacting determinant” (CRD) which is exposed inGPI-anchored proteins that have been hydrolyzed by PIPLC. The antibodyobtained from Oxford GlycoSystems (Wakefield, Mass.), was raised againstthe inositol 1,2 cyclic monophosphate moiety of the trypanosome variantsurface glycoprotein (VSG). Samples were analyzed on 3–11%polyacrylamide gradient SDS gels and transferred to nitrocellulosemembrane. The membrane was blocked in TBS-T (30 mM Tris, 150 mM NaCl, pH7.5, 0.5% Tween 20) containing 5% non-fat dry milk and was incubatedovernight with 4 μg/ml of the anti-CRD antibody at 4° C. The blots werewashed in TBS-T and incubated for three hours at room temperature withthe alkaline phosphatase conjugated secondary antibody (1:1500) (RocheDiagnostics). The membrane was then subjected to chemiluminescenceanalysis (CDP-Star) as detailed by the manufacturers (RocheDiagnostics).

Immunoprecipitation of r150:

Two different anti-CRD antibodies (i) Oxford GlycoSystems antibody and(ii) an antibody raised against a mammalian GPI-anchored pig membranedipeptidase (MDP) were used for immunoprecipitation studies. The latterantibody was isolated from the bulk of the anti-MDP antiserum byfractionation on a column of the immobilized form of the trypanasomevariant surface glycoprotein. Both anti-CRD antibodies are specific tothe inositol 1,2-cyclic monophosphate and have been well characterized(Zamze et al. 1988; Broomfield and Hooper, 1993). Cells were affinitylabeled with 200 pM ¹²⁵I-TGF-β1 and cell extracts were incubated withthe anti-CRD antibody. The resulting immune complexes were treated withprotein A Sepharose (Pharmacia-Biotech) slurry and the beads werepelleted by centrifugation, and were analyzed by SDS-PAGE under reducingconditions followed by autoradiography.

Results

Binding Affinity of r150 for TGF-β Isoforms:

The present inventors have previously reported that in addition to thetypes I, II and III receptors, keratinocytes express a novelGPI-anchored TGF-β1 binding protein r150 which forms a heteromericcomplex with the TGF-β signaling receptors (Tam et al, 1998). Since thisprotein has the potential to regulate TGF-β signaling, it was furthercharacterized. Here was determined the relative affinity of r150 for thethree TGF-β isoforms and, this affinity for TGF-β1 approximates that ofthe TGF-β signaling receptors, which suggests that r150 is predominantlya TGF-β1 binding protein. Keratinocytes affinity labeled with¹²⁵I-TGF-β1 in the absence or presence of increasing concentrations ofunlabeled TGF-β1, -β2 or -β3, were analyzed by SDS-PAGE andautoradiography (FIG. 1 a), and competition curves were created from theautoradiogram for r150, type I and type II receptors using quantitativedensitometry (FIGS. 1 b, c, and d). r150 is not sensitive to reducingagents since its migration pattern is identical when the SDS-PAGEanalysis is done under non-reducing (FIG. 1 a) or reducing conditions(Tam et al, 1998). The half-maximal inhibition of ¹²⁵I-TGF-β1 bindingwas determined from the competition curves as the TGF-β isoformconcentration at which the inhibition was 50% of that observed when nounlabeled ligand was present (Table 1). The concentration of unlabeledTGF-β1 required for half maximal inhibition of ¹²⁵I-TGF-β1 binding byr150 is only 1.2 and 1.3 times higher than that required by type I andII receptors respectively. Although r150 also binds TGF-β3, it does sowith a much lower affinity as compared to the types I and II receptorssince it requires a six-fold higher concentration of TGF-β3 to reachhalf-maximal inhibition of ¹²⁵I-TGF-β1 binding than the types I or IIreceptors. Unlabeled TGF-β2, even at 40 times excess concentrationsminimally inhibited ¹²⁵I-TGF-β1 binding of r150.

Partitioning of the Membrane Bound and Released r150 in Triton X-114:

In order to ascertain that the membrane bound r150 is hydrophobic asexpected of a GPI-anchored protein and that the released r150 behaves asa hydrophilic soluble protein, the temperature dependent phaseseparation property of the non-ionic detergent Triton X-114 was used.Phase separation using Triton-X 114 results in the partitioning ofhydrophilic proteins into the aqueous detergent poor phase whileintegral membrane proteins and lipid attached proteins partition intothe detergent rich phase. This procedure has been useful indistinguishing between the amphipathic (membrane bound) and hydrophilic(released from cell surface) forms of GPI-anchored proteins (Hooper,1992).

Affinity labeled keratinocytes were subjected to Triton X-114partitioning and the detergent rich phase containing hydrophobicproteins and the detergent poor phase containing the hydrophilicproteins were analyzed by SDS-PAGE. As expected of a GPI-anchoredprotein, r150 partitioned predominantly into the detergent rich phase,along with the transmembrane type I, II and III receptors (FIG. 2A). Thepartitioning of soluble r150 in Triton X-114 was then tested. When thedetergent rich phase containing the membrane bound affinity labeled r150was left untreated or treated with PIPLC, and the temperature-inducedphase separation was repeated, the aqueous phase of the sample treatedwith PIPLC was enriched in r150 while that of the sample left untreatedcontained only low amounts of r150 (FIG. 2B). These results stronglyindicate that the PIPLC-released r150 is indeed hydrophilic. Incontrast, the detergent phase of samples left untreated with PIPLCcontained the major portion of r150 while the detergent phase of samplestreated with PIPLC contained minimal amounts of r150 (data not shown).

The Soluble r150 Binds TGF-β1

Was next examined whether r150 released from the cell surface is capableof binding TGF-β1

Data shown in FIG. 3 a demonstrate that soluble r150 in the supernatantobtained from keratinocytes treated with PIPLC could be affinity labeledwith ¹²⁵I-TGF-β1. This binding was specific since it was markedlyreduced when the labeling was done in the presence of unlabeled TGF-β1.unlabeled TGF-β1 did not exhibit any competition for these complexes.

The low molecular weight bands below 97.4 kDa appear to be nonspecificsince unlabeled TGF-β1 did not exhibit competition for these complexesin a reproducible manner. The fact that released r150 binds TGF-β1indicates that r150 is capable of binding the ligand in the absence oftype I, II and III TGF-β receptors or an intact membrane structure.Interestingly, detectable amounts of r150 were observed in thesupernatant not treated with PIPLC, which led us to suspect that theremight be an endogenous phospholipase capable of releasing r150.

That the soluble r150 can inhibit TGF-β1 binding to TGF-β receptors wasdemonstrated using a binding assay. As seen in FIG. 3 b, the supernatantfrom PIPLC treated keratinocytes competed in a dose dependent fashionfor ¹²⁵I-TGF-β as seen by decreased binding to MvLu1 cells. Thesupernatant from a T-25 cm² flask treated with PIPLC inhibited bindingby 33% (p<0.005) and 50% (p<0.04) at doses of 1 and 2 respectively(approximately 1×10⁶ cells, represented as an arbitrary unit of “1” inFIG. 3 b). The inhibition of binding with the supernatant from cells nottreated with PIPLC is consistent with the observation that detectableamounts of r150 is present in this supernatant, alluding to the presenceof an endogenous phospholipase capable of releasing r150 (FIG. 3 a; alsosee below, FIG. 5). This inhibition of ¹²⁵I-TGF-β1 binding to MvLu1cells corresponded to 15%, and 31% (p<0.03 in both cases), respectivelyfor doses 1 and 2. In the PIPLC treated supernatants, the inhibition ofbinding at doses 1 and 2 was significantly higher (p<0.03 in both cases)than in the untreated supernatants.

To rule out the possibility that the competition observed by PIPLCtreated supernatant was due to TGF-β, the supernatant was neutralizedwith anti-TGF-β1 antibody prior to being used in the binding assay.Neutralization with this antibody had no effect on the inhibition by ther150 enriched supernatant (FIG. 3 c). In contrast, 100 pM TGF-β1markedly inhibited ¹²⁵I-TGF-β1 binding and this binding could beneutralized by anti-TGF-β1 antibody but not by non-immune IgG.Furthermore, using a PAI-luciferase assay no TGF-β was detected in thesupernatants of cells untreated or treated with PIPLC (data not shown).Taken together, these results suggest that the released form of r150 iscapable of binding to TGF-β1 and modulating ligand binding to TGF-βreceptors.

Identification of a GPI-Anchor in r150:

Although r150 is sensitive to PIPLC, it is possible that it is notitself GPI-anchored, but is associated with a protein that is GPIanchored. Also, it is conceivable that it is a complex of two lowermolecular weight proteins which became inadvertently cross-linked duringthe affinity labeling procedure. In order to eliminate thesepossibilities, Western blot analysis of r150 was performed after itsrelease from the cell membrane using an anti-CRD antibody specific foran epitope which becomes exposed in GPI anchored proteins only upontreatment with PIPLC.

Keratinocytes were treated with PIPLC and the supernatant was purifiedon a TGF-β1 affinity column. Analysis of fractions by affinity labelingand SDS-PAGE demonstrated that the fraction 21, but not adjacentfractions (represented by fraction 18) contained a 150 kDa TGF-β1binding protein (FIG. 4 a). The binding of ¹²⁵I-TGF-β1 to this proteinis specific since it was blocked in the presence of 5 nM TGF-β1. Theseresults confirm that soluble r150 binds TGF-β1.

When the fractions were analyzed by Western blotting with the anti-CRDantibody, it was revealed that fraction 21, but not other fractionscontained a protein of relative molecular weight of 150 KDa which wasrecognized by the anti-CRD antibody. Detection of a 150 kDa protein bythe anti-CRD antibody in the absence of chemical cross-linkingdemonstrates that r150, but not an associated protein, contains aGPI-anchor and, that r150 does not represent two smaller proteins whichgot inadvertently cross-linked (FIG. 4 b). FIG. 4 b also shows that r150was not detectable in an adjacent fraction (fraction 18).

Evidence to Indicate that an Endogenous Phospholipase C Releases r150 inHuman Keratinocytes:

Next, whether the anti-CRD antibody can immunoprecipitate r150 and/orcoprecipitate the types I and II TGF-β receptors was tested. During thecourse of these studies, it was observed that the anti-CRD antibodyimmunoprecipitated r150 from ¹²⁵I-TGF-β1 labeled keratinocytes, even inthe absence of PIPLC treatment (FIG. 5). This result was reproducibleusing two different anti-CRD antibodies: the Oxford GlycoSystemsantibody that is raised against the CRD epitope of variant surfaceglycoprotein of Trypanosoma brucei (Oxford GlycoSystems), and theantibody specific for the CRD epitope of the porcine membranedipeptidase (MDP, Broomfield and Hooper, 1993). Since both anti-CRDantibodies are specific for the inositol 1,2-cyclic monophosphateepitope exposed only upon PIPLC treatment, recognition by the twoantibodies in the absence of PIPLC treatment indicates that anendogenous phospholipase C cleaved r150 to expose this epitope. Theanti-CRD antibody not only precipitated the r150, but alsocoprecipitated the types I and II receptors. Interestingly, theintensities of the types I and II bands were much stronger than that ofthe r150 itself. The coimmunoprecipitation of the types I and IIreceptors demonstrates the heteromeric complex formation of r150 withthose receptors (FIGS. 5 a, b) which confirms the inventors' previousfinding (Tam et al, 1998). The immunoprecipitation with the anti-CRDantibody is specific because the precipitation of labeled complexes isefficiently blocked when the PIPLC hydrolyzed form of MDP which containsthe epitope to which the antibody was raised against was included in thereaction (FIG. 5 b). These complexes are not detected when the cellswere affinity labeled with ¹²⁵I-TGF-β2 because r150 has a much loweraffinity for TGF-β2 than for the TGF-β1 and TGF-β3 isoforms in thesecells (FIG. 5 c).

Discussion

The present inventors have shown that a novel 150 kDa TGF-β1 accessoryreceptor (r150) forms a heteromeric complex with the TGF-β signalingreceptors on human keratinocytes (Tam et al. 1998). This accessoryreceptor was described as GPI-anchored based on its sensitivity toPIPLC. Here it is demonstrated that the GPI-anchor is contained in r150itself and not on an associated protein and that it binds TGF-β1 with anaffinity similar to those of the types I and II TGF-β receptors. Inaddition, evidence is provided that r150 is released from the cellsurface by an endogenous phospholipase C. The most important finding inthe present work is that the released (soluble) form of r150 bindsTGF-β1 independent of the signaling receptors.

r150 has been characterized as GPI-anchored, based on its sensitivity tophosphatidylinositol specific phospholipase C (PIPLC). In order to provethat the GPI-anchor is present in the r150 itself, it was necessary torule out other possibilities, namely: (i) r150 is not itselfGPI-anchored, but is tightly associated with a protein that isGPI-anchored. Upon PIPLC treatment, the associated GPI-anchored proteinis cleaved which results in the release of both the proteins into thesupernatant. This has been shown to be the case for lipoprotein lipasewhich was initially identified as GPI-anchored protein based on itssensitivity to PIPLC; but it was later found that its PIPLC sensitivitywas a result of close association with a GPI-linked heparan sulfateproteoglycan (Chajek-Shaul et al, 1989). (ii) r150 is a noncovalentlyassociated complex of two lower molecular weight proteins whose combinedmolecular weights equate 150 kDa, of which one component isGPI-anchored. During affinity labeling, the two proteins getinadvertently crosslinked by the chemical crosslinker BS³, and thus uponanalysis by SDS-PAGE, the cross-linked complex is detected at 150 kDa.

By immunoblotting the purified, soluble form of the r150 with theanti-CRD antibody that can specifically recognize the epitope exposed bythe cleavage of the GPI-anchor by PIPLC, the above two possibilitieswere eliminated. Elution from a TGF-β1 affinity column and detection asa 150 kDa protein in the absence of cross-linking, together with itsability to be recognized by the anti-CRD antibody, prove that r150 has arelative molecular weight of 150 kDa and that the GPI-anchor iscontained in r150 itself. There are two GPI-anchored proteins expressedin mammalian tissues that have similar molecular weights as r150. Theseinclude an isoform of NCAM (140 kDa) (Rosen et al, 1992) andceruloplasmin (135 kDa) (Patel and David, 1997). However,immunoprecipitation with antibodies specific to these proteins did notImmunoprecipitate r150 affinity labeled with ¹²⁵I-TGF-β1 (data notshown).

The soluble r150 is capable of binding to TGF-β1 as shown by affinitylabeling of the released protein, and retention on the TGF-β1 affinitycolumn. This suggests that r150 can bind TGF-β in the absence of typesI, II and III receptors or an intact membrane. That soluble r150 has thepotential to modulate TGF-β binding to its receptors was demonstrated byits ability to inhibit ¹²⁵I-TGF-β1 binding to receptors on mink lungcells, Although studies of the inhibition of TGF-β binding to itsreceptors by r150 used cellular supernatant and not purified r150, thisinhibition is most likely due to r150 itself, since there was nomeasurable TGF-β and neutralizing anti-TGF-β antibody had no effect onthis inhibition. The inhibition obtained by the supernatant not treatedby PIPLC is likely due to endogenous release of r150 (FIG. 5). Asexpected, exogenous addition of PIPLC resulted in significantly higherinhibition of binding since more r150 will be released. In addition,r150 is the major TGF-β binder released by PIPLC (FIG. 2). The otherpotential binder, α2-macroglobulin is unlikely to be released insufficient quantity during the one hour incubation.

That soluble r150 binds TGF-β1 is reminiscent of what is observed of theectodomain of type III receptor which has been shown to be released by anot yet characterized mechanism (Lopez-Casillas et al, 1994: Philip etal, 1999). The soluble r150 may act as an antagonist by preventing thebinding of TGF-β to the signaling receptors as has been suggested forthe soluble type III receptor (Lopez-Casillas et al, 1994). This issupported by our finding which suggests that the soluble r150 inhibits¹²⁵I-TGF-β1 binding to receptors on mink lung cells. But unlike the typeIII receptor, r150 would antagonize TGF-β1 activity in an isoformspecific manner, since it has a low affinity for TGF-β2 and a moderateaffinity for TGF-β3, as determined by competition experiments usingunlabeled TGF-β isoforms. Furthermore, affinity cross-link labeling ofkeratinocytes with ¹²⁵I-TGF-β1 or ¹²⁵I-TGF-β3 did not demonstratelabeling of r150 (data not shown). Recently, a soluble type I receptorhas been cloned from a rat kidney cDNA library (Choi, 1999). In contrastto the soluble type III receptor, the soluble type I receptor requiresthe co-expression of the type II receptor in order to bind TGF-β.However, It appeared to potentiate TGF-β signal transduction and theauthor has suggested that this potentiation may be due to thestabilization of the heteromeric TGF-β signaling receptor complex. Theobservation that the soluble r150 can bind the ligand independently ofsignaling receptors, and modulate TGF-β binding to its receptors(present work), together with fact that the membrane bound r150 bindsTGF-β1 and forms heteromeric complex with the type I and II receptors(Tam et al, 1998) raise the possibility that r150 in its membrane boundor soluble form may act as antagonist of TGF-β signaling by regulatingligand availability, or stability of the signaling receptor complex orby directly affecting the signal transduction process.

It is predicted that r150 has dual roles in TGF-β signaling depending onwhether it exists as a cell surface anchored protein or as the solubleform. Consequently, a potential mechanism for the regulation of itsaction would be the hydrolysis of the GPI-anchor. Both the release ofthe protein from the cell surface and the ability of the soluble form tosequester TGF-β may modulate TGF-β receptor function. Such a possibilityin vivo is supported by our observation that an endogenous phospholipaseC releases r150 from the cell surface. Since the anti-CRD antibody onlyrecognizes GPI-proteins released from the cell surface by PIPLC, and thecells were not pretreated with PIPLC, the results indicate that there isan endogenous phospholipase C in keratinocytes capable of hydrolyzingr150 at the same site as the PIPLC. The soluble r150 identified in thecellular extract is not due to protease activity since proteolyticcleavage will result in a protein of lower molecular weight. Neither canit be due to failure of cells to add the GPI anchor during synthesis,since the antibody will not recognize that protein. It is of interest tonote here that r150 was detectable upon overexposure of films in thePIPLC untreated aqueous fractions obtained by Triton X-114 partitioning(data not shown).

Although the presence of a mammalian PIPLC has not been definitivelyestablished, the activity of PIPLC-like enzymes have been implicated inthe insulin signaling pathway in rat liver (Satiel, 1996). Furthermore,Movahedi and Hooper (1997) have demonstrated that the insulin stimulatedrelease of GPI-anchored proteins from differentiated 3T3-L1 adipocytesoccurs via the action of an endogenous phospholipase C. We choose thegeneral term of phospholipase C for the enzyme that releases the r150 inkeratinocytes, since the identity and specificity of the enzyme is notconfirmed. Nevertheless, our results suggest that the release of r150involves an enzyme that hydrolyzes r150 at the same site as PIPLC. PIPLDhydrolyze the GPI-anchor at a different site from PIPLC which does notresult in the formation of the inositol 1,2-cyclic monophosphate, andtherefore the anti-CRD antibodies that we used in this study cannotrecognize PIPLD cleaved proteins (Broomfield and Hooper, 1993). However,PIPLD is expressed in mammalian serum and has been shown to be presentin keratinocytes (Xie et al, 1993). Lin et al. (2002), who describe acell suface antigen named CD109, which appears to be very similar tor150, propose that some GPI anchors are acylated on inositol, because ofCD109 sensitivity to phospholipidase D. It is possible that the activityof both enzymes may be involved in regulating the cell surfaceexpression of r150 on human keratinocytes.

GPI-anchored proteins have been reported to bind TGF-β on certain celllines (Cheifetz and Massague, 1991). More recently, the presentinventors reported the presence of GPI-anchored TGF-β binding proteinson early passage human endometrial stromal cells (Dumont et al, 1995)and human skin fibroblasts (Tam and Philip, 1998). Both endometrialstromal cells and skin fibroblasts displayed a 180 kDa GPI-anchoredTGF-β1 binding protein and a 65 kDa TGF-β2 binding protein. However,whether the GPI anchor is present in the proteins themselves has notbeen ascertained for any of them, and the identities of these proteinsremain unknown. Interestingly, GPI-anchored proteins have beenimplicated in the maintenance of the epidermis. When the expression ofGPI-anchored proteins was abrogated in the skin by the tissue specificdeletion of the PIG-A gene, a gene essential for GPI-protein synthesis,mutants died shortly after birth and their skin was wrinkled and scalyin comparison to that of the wild type (Tarutani et al, 1997). SinceTGF-β has an important role in epidermal homeostasis, it is conceivablethat the GPI-function which is compromised in these mutants is relatedto dysregulated TGF-β action due to the loss of r150.

Our results from the competition experiments demonstrating that r150 hashigh affinity for the TGF-β1 isoform suggest that it is an endogenousligand for this protein. Whether the membrane bound or soluble form ofr150 acts as scavenger receptors regulating ligand availability, whetherthey participate directly in the modulation of downstream signaling, orif the release of the soluble form is a regulated event in vivo, remainto be determined. Identification of its structure should facilitateresolution of these issues. Elucidating the mechanism by which r150functions as an accessory molecule in TGF-β signaling in keratinocytesmay be critical to understanding the molecular mechanisms underlying theregulation of TGF-β action.

EXAMPLE 2 Creation of Keratynocyte Cell Line Mutants Deficient in GPIAnchor Biosynthesis Materials and Methods

Cell Culture: An immortalized human keratinocyte cell line, HaCat, wasobtained from Dr. P. Boukamp (Hedielberg, Germany). HaCat is aspontaneously immortalized cell line which displays no major differencesin differentiation as compared to normal keratinocytes. It possesses atransformed phenotype, but is not tumourigenic (Boukamp et al, 1988).HaCat cells were cultured in D-MEM containing 10% fetal bovine serum, 1mM sodium pyruvate, 2 mM glutamine and 100 U/ml penicillin, 100 μg/mlstreptomycin, and 0.25 μg/ml amphotericin (all Gibco, Burlington,Ontario).

Doubling Time: 8×10⁵ Cells were Seeded at in 60 mm Dishes (Falcon) inDuplicate. At Indicated Times, Cells were Trypsinized and Counted Usinga Hemocytometer

Affinity labeling of cells: Affinity cross-link labeling techniques wereperformed as detailed by Dumont et al (1995). Briefly, monolayers ofcells were washed with ice cold binding buffer [PBS with Ca2+ and Mg2+,pH 7.4 (D-PBS) containing 0.1% BSA] three times over a thirty minuteperiod. Cells were then incubated at 4° C. for three hours with 100 pMof ¹²⁵I-TGF-β1 in the presence or absence of excess non-radioactiveTGF-β1 (Austral Biochemical, Genzyme Inc. or R & D Systemsrespectively). The receptor-ligand complexes were then cross-linked with1 mM Bis(Sulfocsuccinimidyl)suberate (BS3) (Pierce, Rockford Ill.).After 10 minutes, the reaction was stopped by the addition of 500 mMglycine and further incubated for 5 minutes. The cells were then washedtwice with D-PBS and lysed with solubilization buffer (20 mM Tris-HCl,pH 7.4 containing 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 μMphenylmethylsulfonylfloride (PMSF), 200 μg/ml BSA, 1 μg/mL leupeptin, 10μg/ml soyabean trypsin inhibitor, 10 μg/ml benzamide and 2 μg/mlpepstatin). The solubilized material was collected and ⅕ volume of 5×electrophoresis sample buffer (0.25 M Tris-HCl, pH 6.8, 5% SDS, 50%glycerol and trace bromophenol blue) was added. The samples were run ona 1.5 mm-thick 3%–11% SDS-PAGE under nonreducing or reducing (in thepresence of 5% β-mercaptoethanol) conditions. Results were analyzedusing autoradiography followed by quantitative densitometry. [¹⁴C]methylated molecular weight protein markers included myosin (H-chain)(200–220 kDa), phosphorylase-b (97.4 kDa), bovine serum albumin (68kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa),β-lactoglobulin (18.4 kDa) and lysozyme (14.3 kDa) (Gibco orAmersham-Pharmacia Biotech).

Affinity labeling of soluble r150. Confluent monolayers of keratinocyteswere treated with 0.6 U/ml of PIPLC for one hour at 37° C. with mildagitation. The supernatant containing the released GPI-anchored proteinswas collected and concentrated by Centricon 30 (Amicon). To performaffinity labeling of the soluble form of r150, aliquots of theconcentrated supernatant were affinity cross-link labeled with 100–150pM of ¹²⁵I-TGF-β1 in the absence or presence of excess unlabeled TGF-β1as described above except that the solubilization step was omitted.Binding complexes were analyzed by SDS-PAGE and autoradiography.

Isolation and cloning of a keratinoyte cell line mutated in GPI anchorbiosynthesis. The preparation of a keratinocyte cell line mutated in GPIanchor biosynthesis was performed as described by Stevens (1999). HaCatcells grown to 50–60% confluence in a T-75 cm2 flask were treated with300 μg/ml of ethylmethane sulfonate (EMS) for 24 hours at 37° C. in anatmosphere of 5% CO₂/air. EMS is one of the most common chemicalmutagens used to generate cells that are defective in GPI anchorbiosynthesis (Sega, 1984). Cells were then allowed to recover for anadditional 48 hrs. in normal growth medium. Fluorescence activated cellsorting (FACS) was performed to select those cells which lost theexpression of GPI-anchored proteins from their cell surface. Since theidentity of the r150 was not known, GPI anchor deficient cells werenegatively sorted for another GPI-anchored protein, CD59. Briefly, cellswere trypsinized and approximately 2×10⁶ cells were resuspended in onemilliliter of FACS buffer (PBS containing 1% FBS) and incubated with 4μg of fluorescein isothiocyanate (FITC)-conjugated mouse anti human CD59monoclonal antibody (BD Pharmagin, Mississauga, ON.) or mouseFITC-conjugated IgG_(2a) monoclonal antibody for 45 minutes on ice.After the incubation, the cell pellet was washed once and resuspended inone milliliter of FACS buffer. Propidium iodide (3 μg/ml) (Sigma) wasadded to the cells prior to sorting which was performed using the FACSVantage (Becton Dickinson). Cell sorting was repeated two additionaltimes. At the third and final FACS, a single CD59 negative cell wasseeded into each well of a 96 well plate using the FACS Vantageautomated cell deposition unit. In the surviving clones, the cellsurface expression of CD59 was analyzed by flow cytometry to determinewhich cells expressed deficient levels of GPI-anchored proteins.Affinity cross-link labeling with ¹²⁵I-TGF-β1 was performed tocharacterize the expression of the r150.

Luciferase assay. The p3TP-Lux and CMVβ-galectosidase (CMVβB-gal) geneconstructs were gifts from Dr. O'Connor-McCourt (Biotechnology ResearchInstitute, Montreal, Quebec). The p3TP-Lux construct contains threetetradecanoyl phorbol acetate (TPA)-responsive elements and TGF-βresponsive elements from the PAI-1 promoter fused to the luciferasereporter gene (Wrana et al, 1992). Briefly, 2.5×10⁵ HaCat cells, platedin a 12 well plate, were transiently transfected with 1 μg each ofp3TP-Lux and CMVβ-gal cDNAs using the Superfect reagent as recommendedby the manufacturers (Qiagen, Mississauga, Ontario). The cells wereallowed to recover overnight and were serum starved for three to fourhours prior to treatment with various doses of TGF-β1 or TGF-β2 for theindicated times (Austral Biochemicals). For the luciferase assay, cellswere solubilized in 150 μl of lysis buffer (BD Pharmagin, Mississauga,Ontario) for 30 minutes at 4° C. In an opaque 96 well plate, 45 μl oflysate was added to 10 μl of ATP cocktail [0.1 M ATP, 0.5 M KH₂PO₄ (pH7.8), 1 M MgCl₂]. Luciferase activity was measured upon addition of 100μl of 25 mM luciferin (Roche Dagnostics) in 0.1 M KH₂PO₄ (pH 7.8) usingan EG & G Berthold Microplate Luminometer. For the β-galactosidaseassay, in a 96 well plate, 5 μl of the lysate was added to 100 μl ofβ-gal buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 M MgCl₂ and 50mM β-merocaptoethanol) containing 1.5 ng/ml of ortho-nitrophenyl βD-galactopyranoside (ONPG) (Sigma). The samples were incubated at 37° C.until a satisfactory colour reaction was obtained. The colour reactionwas then measured at 420 nm using a spectrophotometric plate reader(Molecular Dynamics, Sunnyvale, Calif.). Values were derived fromtransfections performed in duplicate or triplicate and all experimentswere performed at least three times.

In vitro kinase assay. HaCat and GPI anchor mutated cells grown in T-75cm 2 flasks were serum starved overnight and left untreated or treatedwith 100 pM TGF-β1 for 20 minutes at 37° C. The cells were scraped in200 μl of lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 50 mM NaF, 50 mMβ-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT. 5 mM EDTA pH8.0, 1% NP-40, 10% glycerol, 10 μM PMSF, 200 μg/ml BSA, 1 μg/mLleupeptin, 10 μg/ml soyabean trypsin inhibitor, 10 μg/ml benzamide and 2μg/ml pepstatin) and further incubated in the lysis buffer for 30minutes on ice. The cells were pelleted and the solubilized extract wasprecleared with 40 μl of protein A Sepharose slurry(Amersham-Pharmacia-Biotech) and 3 μg/ml of polyclonal rabbit IgGantibody for two hours at 4° C. One mg of protein was incubated with 3μg/ml of anti-type II TGF-β receptor antibody (Santa Cruz) overnight at4° C. The immune complexes were then incubated with 50 μl of protein ASepharose slurry for two hours at 4° C. The beads were pelleted andwashed three times with 0.5 ml of lysis buffer and one time with 0.5 mlof kinase buffer [50 mM Tris (pH 7.4), 10 mM MgCl₂, 1 mM CaCl₂]. Afterthe last wash, 20 μl of kinase buffer was added to the pellets andincubated with 10 μCi of gamma³²P-ATP (NEN) for 30 minutes at 30° C. Thereaction was halted by the addition of 5 μl of 5× sample buffer (0.25MTris-HCl, pH 6.8, 5% SDS, 50% glycerol and trace bromophenol blue)containing 5% β-mercaptoethanol and boiled for 5 minutes. The extractswere separated on a 7.5% polyacryalamide SDS-PAGE gel and analyzed byautoradiography.

Western blotting of phosphorylated Smad2. HaCat and GPI anchor mutatedcells were grown in 60 mm dishes (Falcon) to 70–80% confluency and serumstarved overnight. Cells were washed with PBS and were treated with withvarious doses of TGF-β1 or TGF-β2 for the indicated times at 37° C. Thecells were solubilized with 500 μl of lysis buffer for at least 30minutes at 4° C. with mild agitation. The cell lysates were collectedand centrifuged for 10 minutes at 12 000×g. The protein concentration ofthe each sample was normalized to 50 μg using the BioRad protein assaykit as recommended by the manufacturers. ⅕ volume of 5× electrophoresisreducing sample buffer was added to the sample and boiled for 5 minutes.The samples were separated on a 7.5% SDS-polyacrylamide gel andtransferred to a nitrocellulose membrane. The membrane was blocked forat least three hours to overnight in blocking buffer [TBS-T buffer: 30mM Tris (pH 7.5), 150 mM NaCl, 0.05% w/v Tween 20, containing 5% w/v ofskim milk powder]. The blot was then incubated for 60 minutes at roomtemperature with the PS2 antibody (1:2000 in TBS-T buffer) whichrecognizes the phosphorylated C-terminus of Smad2 (a gift from Dr. S.Souchelnytskyi, Uppsula, Sweden). The blot was then washed in TBS-Tbuffer and incubated for 45 minutes at room temperature with a secondaryanti-rabbit antibody conjugated to horseradish peroxidase (HRP) (1:5000)(Pierce, Rockford, Ill.). The membrane was then subjected tochemiluminescence analysis (ECL) as detailed by the manufacturers(Amersham-Pharmacia-Biotech). In order to determine equal proteinloading, membranes were immunoblotted with a goat polyclonal anti-Smad2antibody (Santa Cruz) that is raised against a peptide near theN-terminus, or a rabbit polyclonal anti-STAT3 antibody (Santa Cruz)which recognizes a peptide mapping at the C-terminus.

Results

Isolation and Cloning of a Cell Line Mutated in GPI Anchor Biosynthesis:

HaCat cells were treated with 300 μg/ml of ethylmethanesulfonate (EMS)for 24 hours as described in “Materials and Methods.” EMS is anethylating agent that has been used to cause mutagenesis of genes,including those involved in the biosynthesis of the GPI anchor (Sega,1984; Stevens, 1999). Because the identity of the r150 is not known,another GPI-anchored protein that is strongly expressed in humankeratinocytes was used in the cloning of GPI anchor deficient cells.CD59 is a 21 kDa GPI-anchored protein that is expressed in keratinocytesand is an inhibitor of the membrane attack complex (Venneker et al,1994; Pasch et al, 1998). After EMS treatment, cells were stained withan anti-CD59 antibody conjugated to FITC and negatively sorted. Cellsselected into the CD59 negative population were cloned using the FACSVantage automated cell deposition unit. Ten clones survived and werereanalyzed for CD59 expression by flow cytometry. Two clones displayeddecreased expression of CD59 on their cell surface and were presumed tobe mutated in GPI anchor biosynthesis (termed GPI M and GPI M1). FIG. 6Ademonstrates the flow cytometric analysis of parental HaCat cells ascompared to GPI M cells. Though the expression of CD59 was notcompletely abolished in the GPI M cells, the immunofluorescenceintensity of CD59 was analyzed to be approximately half that of HaCatcells (56.3%+/−7.4, p<0.0006). Similar results were obtained with theGPI M1 cells.

In order to ascertain that the GPI anchor mutated cells are deficient inr150, affinity labeling with ¹²⁵I-TGF-β1 was performed. As demonstratedin FIG. 6B, GPI M cells exhibit a significant loss of r150 on their cellsurface as compared to HaCat cells. The expression of the types I, IIand III receptors and their binding to ¹²⁵I-TGF-β1 are unaffected in theGPI M cells and appear identical to that of parental HaCat cells.

Cell Growth and Morphology are Unchanged in GPI Anchor Mutated Cells:

A deficiency or loss of the cell surface expression of GPI-anchoredproteins as a result of EMS mutagenesis is reported not to affect cellmorphology or cell cycle progression (Stevens, 1999). In order toconfirm that cell growth was not altered in the GPI anchor deficientkeratinocyte cell line, the doubling time of these cells was determined.As seen in FIG. 7A, GPI M cells display a similar doubling time(approximately 16 hrs.) as the parental HaCat cells. In addition, thecellular morphology of GPI M cells (FIG. 7C) is unchanged to that of theHaCat (FIG. 7B).

r150 Negatively Modulates TGF-β Stimulated Transcription:

TGF-β1 induced transactivation of the p3TP-Lux reporter construct in GPIM and HaCat cells was then evaluated. As demonstrated in FIG. 8A, upontreatment with 10 pM TGF-β1 for 4 hours, the GPI M cells display anapproximately 30% higher fold increase of luciferase activity, ascompared to HaCat cells (p<0.05). In response to 100 pM TGF-β1, thedifference between the GPI M and HaCat cells rises to 50% (p<0.003).Interestingly, the fold induction in the HaCat cells with 10 pM and 100pM TGF-β1 treatment remain similar to each other (3.3 and 3.9respectively), while the GPI M cells display a dose response,demonstrating fold increases of 4.9 and 8.4 respectively.

Upon 16 hours of TGF-β1 treatment, the GPI M cells display a markedlyenhanced response to 10 pM and 100 pM TGF-β1 compared to the parentalHaCat (FIG. 3B). Also, a dose response to the 10 pM and 100 pM TGF-β1are evident for both cell types. A clone of HaCat cells that wassubjected to the same cloning procedure as the GPI M cells, but notmutated in GPI anchor biosynthesis (GPI NM) was included as a control.Upon 10 pM TGF-β1 treatment, GPI M cells display the highest foldinduction of 21.4 compared to the 13.6 and 10.6 increases demonstratedby the HaCat and GPI NM cells (p<0.005 for GPI M vs HaCat). Similarly,in response to 100 pM TGF-β1, the fold induction exhibited by the GPI Mcells is approximately twice that of HaCat and GPI NM cells (91.2compared to 50.2 and 51.3 respectively) (p<0.01 for GPI M vs HaCat). Inresponse to 100 pM TGF-β2, there is no significant difference in thestimulation of luciferase activity among the GPI M, HaCat and GPI NMcells (FIG. 8C).

GPI Anchor Mutated Cells Display Enhanced Smad2 Phosphorylation at LowDoses of TGF-β1:

In order to determine if the loss of r150 had any affect on theendogenous phosphorylation of Smad2, HaCat and GPI M cells were treatedwith 1–50 pM of TGF-β1 for 20 minutes and immunoblotting was performedwith an antibody raised against the phosphorylated form of Smad2. Asdemonstrated in FIG. 9A, GPI M cells display an enhanced level of Smad2phosphorylation as compared to the parental HaCat. In both cell types,Smad2 phosphorylation becomes detectable at 2 pM TGF-β1 and increases ina dose dependent fashion. However, GPI M cells exhibit an enhanced Smad2phosphorylation in response to low (2 and 5 pM), intermediate (10 pM)and higher (50 pM) doses of TGF-β1 in comparison to the HaCat cells.Immunoblotting with an anti-Smad2 antibody that detects both theunphosphorylated and phosphorylated forms of Smad2 not only demonstratesequivalent protein loading, but shows that the enhanced Smad2phosphorylation is not a result of an increased expression of totalSmad2. Equal protein loading of HaCat and GPI M cell lysates is alsoconfirmed upon immunoblotting with an anti-STAT3 antibody. EnhancedSmad2 phosphorylation in the GPI M cells is not evident in response toTGF-β2, as HaCat and GPI M cells appear to demonstrate the same weakpattern of TGF-β2 induced Smad2 phosphorylation (FIG. 9B).

Elevated Smad2 Phosphorylation is Sustained in GPI Anchor Mutated Cells:

Was further examined if the GPI anchor deficient cells exhibit enhancedSmad2 phosphorylation as compared to HaCat cells upon prolonged exposureto TGF-β1 (FIG. 10). In response to 100 pM TGF-β1 for the indicatedtimes, maximal stimulation of Smad2 phosphorylation in HaCat cells isachieved at 10 minutes and maintained for 180 minutes. In GPI M cells,Smad2 is maximally phosphorylated between 45–90 minutes, followed by amoderate decrease in its phosphorylation at 180 minutes. In comparisonto HaCat cells, the GPI M cells display a significantly elevated Smad2phosphorylation which is evident at 45 minutes and is sustained until180 minutes. The second GPI anchor mutated clone (GPI M1) also exhibitsenhanced Smad2 phosphorylation in response to TGF-β1 (FIG. 10B). In thisexperiment, the GPI M1 cells display an elevated level of TGF-β1 inducedSmad2 phosphorylation as compared to HaCat cells starting at 20 minutesand is sustained until 180 minutes. On the other hand, Smad2phosphorylation in GPI NM control cells is similar to that of HaCatcells for all the time periods studied in response to TGF-β1 (FIG. 10C).Immunoblotting with the anti-STAT3 antibody demonstrates equal proteinloading of the cell lysates. These results indicate that only the GPIanchor deficient cells (GPI M and GPI M1) exhibit enhanced TGF-β1stimulated phosphorylation of endogenous Smad2 as compared to theparental HaCat cells.

Autophosphorylation of Type II Kinase in HaCat and GPI Anchor MutatedCells.

Since r150 can interact with the TGF-β signaling receptors in humankeratinocytes, and TGF-β induced Smad2 phosphorylation is enhanced inGPI anchor deficient cells, we analyzed for alterations in theautophoshorylated state of the constitutively active type II receptorkinase using the in vitro kinase assay. As demonstrated in FIG. 11, GPIM cells do not display any marked differences in type II receptor kinasephosphorylation as compared to the parental HaCat cells in either theabsence or presence of 100 pM TGF-β1. The moderate decrease in TGF-βRIIkinase autophosphorylation displayed by the GPI M cells, as compared toHaCat cells, did not occur in a reproducible manner.

Discussion

A keratinocyte cell line mutated in GPI anchor biosynthesis (GPI M)derived by CD59 negative based FACS from HaCat cells treated with EMS(Stevens, 1999) was created and isolated. In comparison to the parentalHaCat, these cells demonstrate a significant loss in expression of thenovel GPI-anchored TGF-β1 accessory receptor, r150, from their cellsurface. There is no alteration in TGF-β types I and II receptorexpression, nor in their abilities to bind TGF-β1. In addition, GPI Mcells do not show any differences in cellular morphology or doublingtime. However, these cells display an enhanced transactivation of theTGF-β responsive p3TP-Lux reporter gene construct as compared to HaCatcells. Furthermore, GPI M cells display an enhanced Smad2phosphorylation in response to TGF-β1 treatment in a time and dosedependent manner. Taken together, the present work indicates that thenovel accessory receptor, r150 acts as a negative modulator of TGF-βaction in human keratinocytes.

In the isolated GPI anchor deficient cell lines, the cell surfaceexpression of CD59 is still detectable by flow cytometry, thusindicating that the cells do not display a complete abrogation ofGPI-anchored proteins. However, a decrease in immunofluorescenceintensity of approximately 50% exhibited by the GPI M cells iscomparable to the loss of GPI-anchored proteins from the cell surfaceafter PIPLC treatment (Screaton et al, 2000). In addition, ¹²⁵I-TGF-β1affinity cross-link labeling of GPI M cells demonstrate a significantloss of r150 from their cell surface as compared to HaCat cells. ThatGPI anchor biosynthesis is not completely abolished in these cells islikely due to the presence of GPI anchor biosynthetic genes that areresistant to EMS mutagenesis (Stevens et al, 1996). The presentinventors are the first to report of the isolation of a GPI anchordeficient keratinocyte cell line.

EMS is an ethylating agent that has been used to cause mutagenesis ofgenes, including those involved in the biosynthesis of the GPI anchor(Sega, 1984; Stevens, 1999). There is a mutation frequency ofapproximately one in a million cells upon treatment of mammalian cellswith this mutagen in the dose range of 100–400 μg/ml. Hence, to “knockout” two copies of a gene, the frequency becomes 1 000 000×1 000 000(Boyd and Massague, 1989; V. Stevens, personal communication). At thislow frequency, the assumption is made that there is one mutation in eachcell that is selected and cloned. For example, using this methodology,Stevens et al (1996) created a Chinese Hamster Ovary (CHO) cell linemutated in GPI anchor biosynthesis. This cell line has been recentlyused to study the role of GPI-anchored proteins in the development ofAlzheimer's disease (Sambamurti et al, 1999) and in the “cross-talk”between caveolae and GPI-enriched lipid microdomains (Abrami et al,2001). In addition, prior to the cloning of the TGF-β receptors, EMSmutagenesis was used to create the mutant Mv1Lu cell lines known as “R”and “DR” which do not express the type I and types I and II TGF-βreceptors respectively (Boyd and Massague, 1989; Laiho et al, 1990).These TGF-β unresponsive R and DR mutant cell lines continue to be usedto delineate components of the TGF-β signaling pathway (Massague, 1998).Therefore, the main drawback of the above model is that the expressionof all GPI-anchored proteins may be affected. Thus, it is possible thatthe loss of other GPI proteins besides r150 may have an impact on TGF-βsignaling. However, none of the presently identified mammalianGPI-anchored proteins can bind TGF-β or are implicated in TGF-βsignaling (for review, Low, 1989; Turner. 1994). The novel 150 kDaaccessory receptor is the strongest candidate because it appears to bethe only GPI-anchored protein that binds to TGF-β1 in keratinocytes.Furthermore, it has the potential to modulate TGF-β signaling throughits interaction with the types I and II TGF-β receptors. OtherGPI-anchored TGF-β binding proteins such as 180 kDa TGF-β1 bindingprotein and two GPI-anchored TGF-β2 binding proteins at 60 kDa and 140kDa were identified in certain cell lines including a human asteosarcomacell line (Cheifetz and Massague, 1991). In human fibroblasts, thepresent inventors identified a 180 kDa GPI-linked TGF-β1 bindingprotein, as well as a 65 kDa GPI-anchored TGF-β2 binding protein whichdo not interact with the types I and II receptors (Dumont et al, 1995;Tam and Philip, 1998). However, early passage human keratinocytes andHaCat cells do not appear to express any of GPI-anchored TGF-β bindingcomplexes with the above mentioned relative molecular weights (Chapter4; Tam et al, 1998). Together, it is likely that the observeddifferences in TGF-β1 induced cellular responses exhibited by the GPI Mcells is due to the loss of r150.

The GPI anchor deficient cells display an increased transactivation ofthe PAI-1 promoter driven luciferase gene construct upon 4 and 16 hoursof TGF-β1 treatment. The fold induction demonstrated by the GPI M cellsis approximately twice that of HaCat cells and of an isolatedkeratinocyte clone that was not characterized as being mutated in GPIanchor biosynthesis (GPI NM). The conclusion that r150 is a negativemodulator of TGF-β responses is confirmed by enhanced phosphorylation ofendogenous Smad2 exhibited by the GPI anchor deficient cells as comparedto the parental HaCat. This phenomenon is reproducible in both GPIanchor mutants that were cloned (GPI M and GPI M1). In comparison to theHaCat cells. both GPI M and GPI M1 demonstrate a significantly elevatedlevel of Smad2 phosphorylation which is detectable after 20–45 minutesof TGF-β1 treatment and sustained for as long as 180 minutes.Additionally, the GPI M cells demonstrate an enhanced Smad2phosphorylation in response to different doses of TGF-β1 (2–50 μM) ascompared to the parental HaCat cells. Upon treatment with TGF-β2, thereis no detectable alteration in the pattern of Smad2 phosphorylationbetween HaCat and GPI M cells, indicating that the loss of r150 may notimpact TGF-β2 responsiveness. This is also seen in the p3TP-Luxluciferase assay, whereby the HaCat and GPI M cells display similarlevels of TGF-β2 induced luciferase activity. This is not surprisingsince r150 has virtually no affinity for TGF-β2 (Tam et al, 1998). Theincreased intensity and duration of the TGF-β1 induced Smad2phosphorylation likely contributes to the enhanced transactivation ofthe PAI-1 promoter in the GPI anchor deficient cells.

r150's interaction with the types I and II receptors is reminiscent ofanother accessory receptor, endoglin. Endoglin is a 180 kDa homodimerictransmembrane protein that binds TGF-β1 and TGF-β3, and can interactwith the types I and II receptor (see section 1.2.2; Cheifetz et al,1992; Yamashita et al, 1994b). Like the r150, it possesses no kinasedomain, but endoglin displays a short cytoplasmic region that isconstitutively phosphorylated (Yamashita et al, 1994b). Furthermore,overexpression of endoglin exerts an inhibitory effect on TGF-β mediatedgrowth inhibition, PAI-1 induction and angiogenesis (Letamendia et al,1998; Li et al, 2000). However, unlike r150, endoglin cannot bind TGF-βin the absence of the type II receptor and hence, its interaction withthe signaling receptors is a ligand induced phenomenon. Furthermore,endoglin is predominantly expressed in endothelial cells while ourstudies indicate that r150 is not found in endothelial cell types (Wonget al, 2000; Tam and Philip, unpublished observations). Therefore, it ispossible that the mechanisms by which r150 and endoglin exert theirinhibitory effects are distinct from each other.

What are the potential molecular mechanisms by which r150 regulatesTGF-β signaling? Through its interaction with the TGF-β signalingreceptors, r150 may modulate the serine/threonine kinase activity of thetypes I or II receptors. The autophosphorylation of key residues in thetype II receptor is important in the regulation of kinase function (Luoand Lodish, 1997). In vitro kinase assay with the anti-type II receptorantibody indicates that there is no marked difference in the type IIautophosphorylation in GPI anchor deficient cells as compared to that ofHaCat cells in the absence or presence of TGF-β1 (FIG. 11). It ispossible that r150 may interact with the type I receptor to hamper itsphosphorylation by the type II receptor, and hence, activation of thetype I kinase. This would be similar to the action of FKBP12, animmunophilin which interacts with the type I kinase and exerts aninhibitory effect on TGF-β mediated cellular responses (Wang et al,1996). However, our attempts to assess the phosphorylation of the type Ikinase by the in vitro kinase assay with the anti-type I receptorantibody using previously described methods were unsuccessful (Wrana etal, 1994; Wieser et al, 1995). Alternatively, r150's interaction withthe type II receptor may exert a negative regulatory effect on TGF-βsignaling as is seen with TRIP-1, a WD domain protein that specificallyassociated with the type II receptor and suppresses TGF-β inducedtransactivation of the PAI-1 promoter (Choy and Derynck, 1998). However,it is presently unclear if r150 interacts with the signaling receptorsin the absence of TGF-β, or if the interaction is a strictly a ligandinduced phenomenon. In addition, it is unknown if r150 preferentiallyinteracts with the type I or type II receptor.

Our previous work indicates that soluble r150 retains its ability tobind TGF-β1 in absence of the types I and II receptors and of an intactmembrane (Tam et al, 2001). Hence, it is likely that r150 binds toTGF-β1 independently from the TGF-β signaling receptors when attached tothe cell surface. This is in contrast to the type I receptor kinase andendoglin which only recognize TGF-β bound to the type II receptor(Cheifetz et al, 1992; Wrana et al, 1994; Letamendia, 1998). Therefore,the membrane-anchored form of r150 may regulate TGF-β1 binding to itsreceptors. This possibility can be interpreted from the results whichshow that GPI anchor deficient cells display an enhanced Smad2phosphorylation even at low and intermediate doses of TGF-β1 (2–10 pM)as compared to the parental HaCat. Furthermore, in the p3TP-Luxluciferase assay, a dose response is demonstrated by the mutant cellsupon 4 hours of 10 pM (4.9 fold induction) and 100 pM TGF-β1 (8.4 foldinduction) treatment. In contrast, the fold increases in HaCat cells aresimilar at both doses (3.3 and 3.9 respectively). Hence, the access ofTGF-β1 to its receptors appears to be amellorated in the absence ofr150. Therefore, unlike the type III receptor whose role is tofacilitate binding of TGF-β to its signaling receptors, r150 may notplay the role as an “enhance” of TGF-β binding (Lopez-Casillas et al,1993). As a result, the membrane-anchored r150 may act to sequesterTGF-β1, but not TGF-β2 or TGF-β3, away from the signaling receptors onthe call surface. Thus, in the absence of r150. Lower amounts of TGF-β1can induce the heteromerization of the types I and II receptors whichcan surpass the signaling threshold required to activate the type Ikinase, leading to Smad2 phosphorylation.

r150 may potentially have a role in the cellular “compartmentalization”of the types I and II receptors and of the R-Smads. There is an emergingtheme in signal transduction biology whereby signaling molecules can becompartmentalized into membrane entities known as caveolae and “lipidrafts.” These are organized lipid microdomains which as serve centres inwhich signaling molecules of various pathways can effectively interact(Sargiacomo et al, 1993; Horejsi et al, 1999). GPI-anchored proteinshave been detected in caveolae or lipid rafts in association with othersignaling molecules such as Src-like kinases, G-proteins, PKC, and PDGFreceptor (Sargiacomo et al, 1993; Lisanti et al, 1994; Oka et al, 1997;Liu et al, 1997). The association of r150, a GPI-anchored protein, withthe types I and II receptors suggests that the TGF-β receptors may alsobe organized in these plasmalemmal entities. This is a possibility sincecaveolin-1, the main scaffolding protein in caveolae, was recentlyreported to co-localize with the types I and II receptors and Smad2 incaveolae-enriched membrane fractions. The type I receptor wasdemonstrated to directly associate with caveolin-1 which resulted in thedownregulation of TGF-β mediated transcriptional responses andsuppression of ligand induced Smad2 phosphorylation (Razani et al,2001). Furthermore, the FYVE domain in Smad anchor for receptoractivation (SARA), a protein that plays a prominent role in controllingthe subcellular localization of Smad2, was required to maintain SARAlocalized in punctate “spots” near the cell surface as characterized byimmunofluorescence microscopy (Tsukazaki et al, 1998). SARA was shown torecruit Smad2 into these domains where TGF-β receptors alsoco-localized. The identity of these “spots” were not confirmed, but arespeculated to be lipid rafts. Presently, it is not known whether SARAinteracts with r150. That SARA and r150 may synergistically act tolocalize r150 into lipid microdomains is an interesting possibility.within caveolae or lipid rafts, the TGF-β signaling machinery may alsopotentially interact with signaling molecules of other pathways thatreside in these structures. This would provide a structural explanationas to how TGF-β can elicit the participation of multiple signalingpathways.

GPI-anchored proteins are implicated in the maintenance of skinhomeostasis. Targeted deletion of the GPI anchor biosynthetic gene,PIG-A, in the epidermis of transgenic mice results in smaller pupsexhibiting skin with a wrinkled appearance and thickened stratum corneumas compared to wild type mice (Tarutani et al, 1997). This defect inskin development is the apparent cause of death of these mice within 1–3days after birth. Interestingly, transgenic mice engineered tooverexpress TGF-β1 in the epidermis also demonstrate a compact stratumcorneum with an increase in the stratified cell layers as compared towild type mice (Sellheyer et al, 1993). Due to the overexpression ofTGF-β1, there is a significant reduction in the number of proliferatingepidermal cells as determined by pulse labelling with5-bromodeoxyuridine. Death of these transgenic mice is also attributedto abnormal skin development. Taken together, the targetedoverexpression of TGF-β1 in the epidermis appears to mimic that of thePIG-A deletion. It is possible to envision that the ablation of r150'sexpression in the epidermis as a consequence of the PIG-A deletion wouldresult in the loss of its inhibitory effects in TGF-β action, thuscontributing to the subsequent hyperactivity of TGF-β1 signaling in theskin. Hence, r150 may play an essential role in skin development as akey regulator of TGF-β's function in epidermal differentiation andhomeostasis.

In conclusion, studies of TGF-β responses in the GPI anchor deficientkeratinocytes have provided critical insight into r150's function inTGF-β signaling. The present results indicate that r150 negativelymodulates TGF-β action in human keratinocytes. In r150 deficientkeratinocytes, TGF-β induced Smad2 phosphorylation and PAI-1 expressionare enhanced. It is conceivable that r150 may directly modulate type Iand II kinase activity through its interaction with the signalingreceptors. In addition, due to r150's ability to bind TGF-β1 on its own,the membrane bound and soluble r150 may regulate ligand availability byacting as a scavenger receptor. Delineation of r150's structure isnecessary to elucidate the precise molecular mechanisms by which thisnovel accessory receptor regulates TGF-β signaling.

EXAMPLE 3 Sequence of r150 and Nucleic Acids Encoding the Same

Cloning of r150 and expression of r150 gene confirm that r150 is aninhibitor of TGF-responses: To determine its structural identity, r150was purified on a TGF-1 affinity column and analyzed by tandem massspectrometry (Harvard Microchemistry Facility, Harvard Univ) whichallowed us to obtain a 19 amino acid microsequence. This matched to the5′ end of an express sequence tagged (EST) cDNA clone of unknownfunction from human placenta. Subsequently, sequencing by PCR usingseven primers, the full sequence was obtained. Alignment and conserveddomain analysis revealed it to be a novel protein of 1428 amino acidswith an N-terminal signal sequence and a C-terminal GPI anchorattachment signal sequence (FIG. 12 and SEQ ID No: 2). The most likelyGPI anchor cleavage site (ω) is at amino acid residue 1404. Thepredicted molecular mass and structural features indicate that this geneproduct represents r150. This novel TGF-accessory receptor has athiolester signature motif and belongs to the complementC3/2-macroglobulin superfamily as recently found in Lin et al, (2002).These authors, using a very different strategy (affinity binding ofblood cells types with monoclonal antibodies) have identified a proteinwhich has 1445 amino acids, which is very similar to r150. No definitefunction has been assigned to this protein called CD109, considered bythe present inventors to be a variant of r150. Expression studies inHaCaT and 293 cells confirm that the cDNA encodes a 150 kDa GPI-anchoredprotein.

Transfection of HaCaT and 293 cells with the r150 gene (SEQ ID NO: 1)and detection of the expressed protein by an antibody specific for GPIanchor (anti-CRD antibody) demonstrate that the expressed proteinmigrates at 150 kDa and that it contains a GPI anchor as expected ofr150 (FIG. 13). Furthermore, overexpression of r150 in HaCaT and 293cells results in strong negative modulation of TGF-induced Smad 2phosphorylation and gene promoter activity, providing strong evidence toconfirm that r150 is an inhibitor of TGF-signaling and responses. Cellstransfected with r150 displayed markedly decreased Smad 2phosphorylation upon stimulation with TGF- when compared withuntransfected cells or cells transfected with the empty vector (FIG.14). Similarly, transfection of cells with the 3TPLUX promoter-reporterconstruct (encoding plasminogen activator inhibitor promoter linked tothe luciferase reporter gene) resulted in a marked decrease in both thebasal and TGF-induced promoter activity (FIG. 15). Taken together, ourresults demonstrate that r150 is an inhibitor of TGF-signaling andresponses in vitro and implicate r150, in its membrane anchored and/orsoluble form, as a key regulator TGF-action in vivo (FIG. 16).

Sequence comparisons with the ones disclosed by Lin et al (2002) andSchuh et al. (2002) provide indications of variants (see FIG. 17). Someamino acid residues may change, but would presumably not change theproperty of binding TGF-β1. At least two types of r150-like would exist:one with Tyr, one with Ser at position 703. The TGF-β1 binding regioncan be predicted by sequence comparison with the sequences disclosed forα₂ macroglobulin (Webb et al. 1998). The TGF-β1 binding domain isascribed between amino acids 591 and 774 of α₂-M protein sequence. Theminimal binding sequence appears to be the 16-mer WDLWUNSAGVAEVGU (Webbet al. 2000). r150 corresponding sequence shows little homology withthis sequence. However, the surrounding sequences are more homologous tor150. Besides that, r150 appears to be much more selective than α₂M forTGF-β1. It is therefore possible that the above 16-mer is sort of a“consensus” sequence with allows versatility in the binding of aplurality of cytokins. The corresponding r150 sequence would be muchmore specific to TGF-β1. This sequence is SEQ ID No: 12 for r150 and itscoding nucleic acid is defined in SEQ ID NO: 11. Any protein comprisingthis particular minimal 16mer is within the scope of this invention.

EXAMPLE 4 Mapping the TGF-β Binding Domain of r150

To confirm that r150 binding domain corresponds to th one found for α₂M,the following procedure is performed.

The ligand binding domain of r150 is mapped by producing deletionmutants of r150 and analyzing ligand binding activity. The requirementfor the GPI anchor for r150 function is examined using chimericconstructs in which the C-terminal GPI anchor sequence of r150 isreplaced with the transmembrane domain (TM) or the GPI anchor sequenceof an irrelevant protein, and determining alterations in TGF-βresponses. The deletion mutagenesis and creation of chimeric constructsis done as described previously in collaboration with Dr. Uri Saragovi,McGill. (Taheri et al, 2000; Zaccaro et al, 2001)

Deletion mutants are generated using progressive digestion of r150 cDNAusing BAL-31 exonuclease starting ˜20 amino acids down stream from thesignal peptide. Digests are repaired and the fragments are sub-cloned toreattach the signal peptide. The deletion mutants, the full length r150or the empty vector are expressed in COS-7 cells and in GPI mutantkeratinocytes as previously described (Pepin et al, 1994). Cells willthen be affinity labeled with 125I-TGF-β1 and analyzed by SDS-PAGE todetermine ligand binding activity as described previously (Tam andPhilip, 1998). The generation of chimeric constructs (r150 in which itsGPI anchor sequence is replaced with the TM of insulin receptor or withthe GPI anchor sequence of NCAM), was performed using overlapping PCR aspreviously described (Screaton et al, 2000; Zaccaro et al, 2001). Wildtype r150 and the chimeric constructs are expressed in COS-7 cells andin GPI mutant keratinocytes, and TGF-β responses are determined.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

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1. A method for inhibiting TGF-β1 activity in a biological tissue of ananimal comprising an administration thereto of an effective amount of aprotein comprising a sequence selected from the group consisting of: a)SEQ ID NO:2 b) amino acids 694–712 of SEQ ID NO:2; c) amino acids651–683 of SEQ ID NO:2 d) a protein sequence having a tyrosine atposition 703 of SEQ ID NO:2; e) a protein sequence having amino acids 21to 1428 of SEQ ID NO:2 f) a protein sequence having amino acids 21 to1404 of SEQ ID NO:2; and g) a protein sequence having a methionineinstead of threonine at position 1224 of SEQ ID NO:2 thereby inhibitingTGF-βl activity in a biological tissue of an animal.
 2. The method ofclaim 1, wherein said protein has the amino acid sequence of SEQ IDNO:2.